Production of myristate in plant cells

ABSTRACT

By this invention, methods to produce C14 fatty acids in plant seed oils are provided. In a first embodiment, this invention relates to particular C14 preferring acyl-ACP thioesterase sequences from Cuphea palustris, camphor and nutmeg, and to DNA constructs for the expression of these thioesterases in host cells for production of C14 fatty acids. Other aspects of this invention relate to methods for using other plant medium-chain thioesterases or medium-chain thioesterases from non-plant sources to provide C14 fatty acids in plant cells. In this regard, the production of C14 fatty acids in plant cells as the result of expression from an elm medium chain acyl-ACP thioesterase and a bacterial luxD gene is provided.

This application is a continuation-in-part of U.S. Ser. No. 08/383,756filed Feb. 2, 1995, issued as U.S. Pat. No. 5,654,495, and acontinuation-in-part of U.S. Ser. No. 07/968,971 filed Oct. 30, 1992,issued as U.S. Pat. No. 5,455,167, and a continuation-in-part ofPCT/US93/10814 filed Oct. 29, 1993, and a continuation-in-part of U.S.Ser. No. 08/261,695, filed Jun. 16, 1994 now abandoned.

TECHNICAL FIELD OF THE INVENTION

The present invention is directed to nucleic acid sequences andconstructs, and methods related thereto.

INTRODUCTION BACKGROUND OF THE INVENTION

Members of several plant families synthesize large amounts ofpredominantly medium-chain (C8-C14) triglycerides in specialized storagetissues, some of which are harvested for production of important dietaryor industrial medium-chain fatty acids containing oils (F. D. Gunstone,The Lipid Handbook (Chapman & Hall, New York, 1986) pp. 55-112). Lauricoil (those containing C12:0 fatty acyl groups) and its derivatives findwidespread use, particularly in the soap, detergent and personal careindustries.

Over the past several years, mildness has become increasingly importantin differentiating soaps, detergents and personal care products, with anemphasis on developing surfactants that combine acceptable performancewith improved mildness. Myristate (C14:0) based surfactants offer anexcellent combination of cleansing and mildness. However, limitations onthe supply of myristate have precluded significant use of thesesurfactants, despite their functional superiority in certainapplications. Myristate is available only in relatively small quantitiesas a coproduct of the fractionation of lauric oils. Coconut oil containsapproximately 48% C12:0 and 17% C14:0, and palm kernel oil containsapproximately 51% C12:0 and 18% C14:0. Only a fraction of the C14:0present in these oils, however, is available as purified C14:0(myristate), as most commercial "lauric fatty acid/methyl ester"products contain significant amounts of myristate, in addition to theprimary laurate component. Thus, myristate based derivatives currentlyfind only limited use in the personal care product industry due to thehigh cost involved in their production.

LITERATURE

Pollard, et al., (Arch. of Biochem. and Biophys. (1991) 284:1-7)identified a medium-chain acyl-ACP thioesterase activity in developingoilseeds of California bay, Umbellularia californica. The baythioesterase was subsequently purified by Davies et al., (Arch. Biochem.Biophys. (1991) 290:37-45) which allowed the cloning of a correspondingcDNA which has been used to modify the triglyceride composition ofplants(WO 91/16421 and WO 92/20236).

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E. The nucleic acid sequence and translated amino acidsequence of Cuphea palustris C14:0-ACP thioesterase cDNA clone (SEQ IDNO:1) MCT34 (CpFatB2) are provided.

FIGS. 2A-2E. The nucleic acid sequence and translated amino acidsequence of a nutmeg (Myristica fragrans) Class II type thioesterase,MYRF-1 (MfFatB2) (SEQ ID NO:2), having preferential activity onC14:0-ACP is provided.

FIGS. 3A-3E. The nucleic acid sequence and translated amino acidsequence of a nutmeg (Myristica fragrans) Class II type thioesterase,MYRF-2 (MfFatB1) (SEQ ID NO:3), having preferential activity onC14:0-ACP is provided.

FIGS. 4A-4C. Nucleic acid and translated amino acid sequence (SEQ IDNO:4) of a PCR fragment containing the encoding region for the matureprotein portion of a camphor Class II acyl-ACP thioesterase is provided.

FIGS. 5A-5D. The nucleic acid sequence and translated amino acidsequence of an elm acyl-ACP thioesterase partial cDNA clone (SEQ IDNO:5) are provided.

FIGS. 6A-6B. The nucleic acid sequence of a Cuphea hookeriana CUPH-4thioesterase cDNA clone (SEQ ID NO:6), CMT13, is provided.

FIGS. 7A-7C. Nucleic acid sequence (SEQ ID NO:7) of an oleosinexpression cassette is provided.

FIG. 8. Mole % fatty acid composition data from single seeds of Brassicaplants 3854-3 and 3854-11, expressing a nutmeg FatB thioesterase, areprovided.

FIG. 9A-9B. Mole % fatty acid composition data from single seeds ofBrassica plants 5233-5 (FIG. 9A) and 5233-6 (FIG. 9B), expressing acamphor FatB thioesterase, are provided.

FIG. 10A-10C. Mole % fatty acid composition data from single seeds ofBrassica plants 3863-10, 3863-7, 3863-4, 3863-8, 3863-2 and 3863-5expressing a C. palustris FatB thioesterase, are provided.

SUMMARY OF THE INVENTION

By this invention, plant genes encoding acyl-ACP thioesterases havingthe ability to act on C14:0-ACP substrate to form free C14:0 (myristate)are provided. The invention encompasses sequences which encodebiologically active thioesterases from plants or bacteria, as well assequences which are to be used as probes, vectors for transformation orcloning intermediates. Biologically active sequences are preferentiallyfound in a sense orientation with respect to transcriptional regulatoryregions found in various constructs. The instant invention pertains tothe entire or portions of the genomic sequence or cDNA sequence and tothe thioesterase protein encoded thereby, including precursor or matureplant thioesterases.

Various plant genes encoding thioesterases having the ability tohydrolyze C14:0-ACP substrate are exemplified herein, and may beobtained for example from Cuphea species, nutmeg, camphor and elm. Theexemplified plant thioesterase sequences may also be used to obtainother related plant thioesterase genes.

Other sources of genes which encode proteins capable of hydrolyzing C14acyl-ACP substrates are also considered herein, and in particular, C14acyl-ACP thioesterase activity of a luxD gene obtainable frombioluminiscent bacteria is demonstrated. Constructs and methods forproducing C14:0 in host cells using a luxD gene from Vibrio harbeyi areprovided herein.

Of special interest are recombinant DNA constructs which can provide forthe transcription or transcription and translation (expression) of thedisclosed protein sequences. In particular, constructs which are capableof transcription or transcription and translation in plant host cellsare preferred. Such construct may contain a variety of regulatoryregions including transcriptional initiation regions obtained from genespreferentially expressed in plant seed tissue.

In a second aspect, this invention relates to the presence of suchconstructs in host cells, especially plant host cells, and to a methodfor producing proteins having C14 acyl-ACP thioesterase activity in ahost cell or progeny thereof via the expression of a construct in thecell. In a related aspect, this invention provides transgenic host cellswhich have an expressed protein having C14 acyl-ACP thioesteraseactivity therein.

In a different embodiment, this invention relates to methods of using aDNA sequence encoding a protein having hydrolysis activity on C14:0acyl-ACP substrates for the modification of the proportion of fattyacids produced within a cell, especially plant cells. Plant cells havingsuch a modified fatty acid composition are also contemplated herein.

Of particular interest is the modification of the fatty acid compositionof storage triglycerides in oilseed, plants for increased proportion ofC14:0 fatty acyl groups. In this manner, seeds with modified oils havingnovel fatty acyl compositions are produced. Such novel seeds and oilsare also encompassed by the instant invention.

DETAILED DESCRIPTION OF THE INVENTION

A protein capable of hydrolyzing C14 acyl-ACP substrates for use in theinstant invention includes any sequence of amino acids, peptide,polypeptide or protein which demonstrates the ability to catalyze theproduction of free fatty acid(s) from C14:0-ACP substrates under plantenzyme reactive conditions. By "enzyme reactive conditions" is meantthat any necessary conditions are available in an environment (i.e.,such factors as temperature, pH, lack of inhibiting substances) whichwill permit the enzyme to function. Such proteins having C14 hydrolysisactivity may be acyl-ACP thioesterases obtainable from plant sources, ormay be from other sources, such as bacteria.

Of particular interest in the instant application are plant acyl-ACPthioesterases which have hydrolysis activity primarily on for C14:0-ACPsubstrates as compared to other acyl-ACP substrates, including medium-or long-chain acyl-ACP substrates. In this regard, thioesterase encodingsequences obtainable from Cuphea palustris are of particular interest inthe instant invention. Other plant thioesterases having C14:0-ACPactivity are also of interest, so long as the thioesterase demonstratespreferential activity on C14:0-ACP substrates, as compared to othermedium-chain acyl-ACP substrates, i.e. those having carbon chain lengthsof C8, C10 or C12. Thus, acyl-ACP thioesterases from nutmeg and camphor,which have substantial activity on C14:0-ACP substrates, as well as someactivity on longer and other medium-chain substrates, are alsoencompassed by the instant invention. Also considered useful forproduction of C14 fatty acyl groups in plant seeds is a medium-chainacyl-ACP thioesterase from elm which demonstrates hydrolysis activitymainly on C10 and C16 substrates upon expression in E. coli, but resultsin increased levels of C14:0 fatty acids when expressed in E. coli andincreased levels of C14:0 fatty acyl groups in triglycerides whenexpressed in transgenic plant seeds. Thus, it is recognized that plantacyl-ACP thioesterases useful for C14 production may also demonstratehydrolysis activity on longer chain acyl-ACP substrates, such as thosehaving carbon chain lengths of C16 or C18.

Also of interest in the instant application are proteins from non-plantsources which may be demonstrated to have acyl-ACP thioesterase activityon C14:0 acyl-ACPs, for example bacterial proteins having a primaryenzymatic activity of an acyl-transferase, but which can be demonstratedto be capable of acyl-ACP hydrolysis activity on C14:0-ACP substrates.Of particular interest as sources of bacterial genes which encodeproteins having C14 acyl-ACP activity are bioluminescent bacteria. Asdemonstrated herein for the bacterium Vibrio harveyi, the luxD genes insuch bacteria provide a desired C14:0-ACP thioesterase activity.

In addition to the plant C14:0-ACP thioesterase sequences exemplifiedherein, acyl-ACP thioesteraes from other plant species are also ofinterest in the instant invention. Target plant species for isolation ofgenes encoding thioesterase having activity on C14:0-ACP substratesinclude those which have been reported to accumulate significant levelsof C14 fatty acids, such as Myristicaceae, Simarubaceae, Vochysiaceae,and Salvadoraceae, and rainforest species of Erisma, Picramnia andVirola. For isolating C14:0-ACP thioesterase genes, nucleic acid probesmay be prepared from C14:0-ACP thioesterase sequences provided herein,or from other plant medium-chain acyl-ACP thioesterase sequences whichhave been described.

Plant thioesterases, including medium-chain plant thioesterases aredescribed in WO 91/16421 (PCT/US91/02960) and WO 92/20236(PCT/US92/04332), which are hereby incorporated by reference in theirentirety. Analysis of the encoding sequences and translated amino acidsequences of a number of plant acyl-ACP thioesterases has demonstratedthe existence of two evolutionary classes of plant acyl-ACPthioesterases which are designated as "Class I" or "FatA" (for fattyacyl transferase type A) and "Class II" (or "FatB"). These classes arenot a simple reflection of phylogenetic relationships of the variousplants from which the thioesterase encoding sequences were obtained. Forexample, a Cuphea hookeriana FatA clone (clone CLT7 in FIG. 10 of WO94/10288) is closely related to safflower FatA clones (sequencesprovided in FIG. 4 of WO 92/20236). In contrast, a Cuphea hookerianaFatB clone (CUPH-1 clone in FIG. 6 of WO 94/10288) is equally distant inevolutionary relationship from the Cuphea hookeriana FatA clone and thesafflower FatA clone.

Class I thioesterases have been found in mango (FIG. 1), safflower,Brassica campestris and Cuphea hookeriana, which sequences are providedin U.S. Ser. No. 07/949,102, filed Sep. 21, 1992, now pending, and in WO92/20236 and WO 94/10288. The plant Class I type thioesterases whichhave been described to date have preferential activity on longer chainacyl-ACP substrates, particularly 18:1-ACP. Class II thioesterases havebeen discovered in California bay, elm, Cuphea hookeriana, Arabidopsisthaliana and camphor. The plant C14:0 acyl-ACP thioesterases describedherein are also of the Class II type. All medium-chain preferringacyl-ACP thioesterases described to date, including those havingactivity on C14:0, are of the Class II type. Thus, additional plantacyl-ACP thioesterases having activity on C14:0 substrates may beidentified through sequence homology to medium-chain acyl-ACPthioesterases.

For example, a C. palustris C14 acyl-ACP thioesterase exemplified hereinwas obtained by screening a gene library with encoding sequences formedium-chain preferring acyl-ACP thioesterases from Cuphea hookeriana.Although the C. hookeriana gene sequences encode thioesterases havingpreferential activity on C8, C10 or C16 fatty acids, the substantialsequence homology within thioesterase genes in various Cuphea speciesallowed for detectable hybridization of the C palustris C14 clone to theC hookeriana gene probes. For hybridization of C14 thioesterases fromplants other than Cuphea species, direct hybridization techniques mayalso be successful under low stringency conditions. For example, nutmegC14:0-ACP thioesterase clones described herein were obtained by lowstringency hybridization screening using a bay C12:0-ACP thioesterasegene fragment as probe. Thus, medium-chain acyl-ACP thioesterase genesfrom other plant species may be used to identify C14 acyl-ACPthioesterase genes. In addition, highly conserved regions have beenidentified in various plant medium-chain thioesterase amino acidsequences. Such regions find particular use in identification ofadditional medium-chain thioesterase genes, including those havingpreferential activity on C14:0-ACPs, for example by PCR amplificationtechniques.

As noted above, plants having significant presence of C14:0 fatty acidstherein are preferred candidates to obtain naturally-derived C14:0 plantthioesterases. However, it should also be recognized that other plantsources which do not have a significant presence of C14:0 fatty acidsmay be screened as additional enzyme sources. For example, as discussedherein, a camphor acyl-ACP thioesterase gene was discovered to havepreferential hydrolysis activity on C14:0-ACP substrates, with onlyminor activity on C12:0-ACP substrates, although analysis of camphorseed oil composition indicates significant levels of C12:0 fatty acylgroups and only low levels of C14 fatty acids. Thus, expression ofmedium-chain acyl-ACP thioesterases in E. coli may be used to identifyacyl-ACP thioesterases which find use in production of C14:0 fatty acidsin transgenic plant seed oils.

Northern analysis of candidate plant acyl-ACP thioesterase genes mayalso be useful to identify those having activity on C14:0 fatty acids.In Cuphea hookeriana, a clone, CUPH-1, which is expressed at low levelsin various plant tissues has been demonstrated to have hydrolyticactivity primarily on 16:0 acyl-ACP substrates. A related C. hookerianathioesterase clone, CUPH-2, however, was demonstrated to be highlyexpressed and seed specific. This CUPH-2 clone was found to havehydrolytic activity primarily on medium-chain acyl-ACP substrates,namely C8 and C10. Similarly, C. hookeriana CUPH-4 is highly expressedin a seed specific manner, and as demonstrated further in the Examplesherein, may be used to provide for increased production of C14 fattyacids in transformed host cells.

One skilled in the art will readily recognize that antibodypreparations, nucleic acid probes (DNA and RNA) and the like may beprepared and used to screen and recover "homologous" or "related"thioesterases from a variety of plant sources. For immunologicalscreening methods, antibody preparations either monoclonal or polyclonalare utilized. For detection, the antibody is labeled using radioactivityor any one of a variety of second antibody/enzyme conjugate systems thatare commercially available. Examples of some of the available antibodydetection systems are described by Oberfilder (Focus (1989) BRL LifeTechnologies, Inc., 11:1-5).

For nucleic acid screening methods, genomic or cDNA libraries preparedfrom a candidate plant source of interest may be probed with conservedsequences from plant thioesterase to identify homologously relatedsequences. Homologous sequences are found when there is an identity ofsequence, which may be determined upon comparison of sequenceinformation, nucleic acid or amino acid, or through hybridizationreactions between a known thioesterase and a candidate source.Conservative changes, such as Glu/Asp, Val/Ile, Ser/Thr, Arg/Lys andGln/Asn may also be considered in determining amino acid sequencehomology. Amino acid sequences are considered homologous by as little as25% sequence identity between the two complete mature proteins. (Seegenerally, Doolittle, R. F., OF URFS and ORFS (University Science Books,Calif., 1986.)

Typically, a lengthy nucleic acid sequence may show as little as 50-60%sequence identity, and more preferably at least about 70% sequenceidentity, between the target sequence and the given plant thioesteraseof interest excluding any deletions which may be present, and still beconsidered related. When longer nucleic acid fragments (>100 bp) areemployed as probes, such as large cDNA fragments, one may screen withlow stringencies (for example 40°-50° C. below the melting temperatureof the probe) in order to obtain signal from the target sample with20-50% deviation, i.e., homologous sequences. (See, Beltz, et al.Methods in Enzymology (1983) 100:266-285.).

Shorter probes are also useful in thioesterase gene isolationtechniques, and find particular applications in polymerase chainreactions (PCR). As described in more details in the following examples,medium-chain thioesterase gene fragments may be obtained by PCR usingprimers to sequences which are highly conserved in plant medium chainacyl-ACP thioesterase protein sequences.

Using methods known to those of ordinary skill in the art, a DNAsequence encoding a protein having hydrolytic activity on C14:0-ACPsubstrate can be inserted into constructs which may then be introducedinto a host cell of choice for expression of the enzyme, including plantcells for the production of transgenic plants. Thus, potential hostcells include both prokaryotic and eukaryotic cells. A host cell may beunicellular or found in a multicellar differentiated or undifferentiatedorganism depending upon the intended use. Cells of this invention may bedistinguished by having a protein having hydrolysis activity on C14:0acyl-ACP substrates foreign to the wild-type cell present therein, forexample, by having a recombinant nucleic acid construct encoding a plantthioesterase therein.

Also, depending upon the host, the regulatory regions will vary,including regions from viral, plasmid or chromosomal genes, or the like.For expression in prokaryotic or eukaryotic microorganisms, particularlyunicellular hosts, a wide variety of constitutive or regulatablepromoters may be employed. Among transcriptional initiation regionswhich have been described are regions from bacterial and yeast hosts,such as E. coli, B. subtilis, Sacchromiyces cerevisiae, including genessuch as betagalactosidase, T7 polymerase, tryptophan E and the like.

For the most part, when expression in a plant host cell is desired, theconstructs will involve regulatory regions (promoters and terminationregions) functional in plants. The open reading frame, coding for theprotein having hydrolytic activity on C14:0-ACP substrate will be joinedat its 5' end to a transcription initiation regulatory region such asthe wild-type sequence naturally found 5' upstream to a plantthioesterase structural gene. Numerous other transcription initiationregions are available which provide for a wide variety of constitutiveor regulatable, e.g., inducible, transcription of the structural genefunctions. Among transcriptional initiation regions used for plants aresuch regions associated with the structural genes such as for CaMV 35Sand nopaline and mannopine synthases, or with napin, ACP promoters andthe like. The transcription/translation initiation regions correspondingto such structural genes are found immediately 5' upstream to therespective start codons. If a particular promoter is desired, such as apromoter native to the plant host of interest or a modified promoter,i.e., having transcription initiation regions derived from one genesource and translation initiation regions derived from a different genesource, including the sequence encoding the plant thioesterase ofinterest, or enhanced promoters, such as double 35S CaMV promoters, thesequences may be joined together using standard techniques. For mostapplications desiring the expression of C14:0-ACP thioesterases inplants, the use of seed specific promoters is preferred.

When expression of the proteins of the instant invention is desired inplant cells, various plants of interest include, but are not limited to,rapeseed (Canola varieties, including low linolenic lines, and HighErucic Acid varieties), sunflower, safflower, cotton, Cuphea, soybean,peanut, coconut and oil palms, and corn. Depending on the method forintroducing the recombinant constructs into the host cell, other DNAsequences may be required. Importantly, this invention is applicable todicotyledyons and monocotyledons species alike and will be readilyapplicable to new and/or improved transformation and regulationtechniques.

In any event, the method of transformation is not critical to theinstant invention; various methods of plant transformation are currentlyavailable. As newer methods are available to transform crops, they maybe directly applied hereunder. For example, many plant species naturallysusceptible to Agrobacterium infection may be successfully transformedvia tripartite or binary vector methods of Agrobacterium mediatedtransformation. In addition, techniques of microinjection, DNA particlebombardment, electroporation have been developed which allow for thetransformation of various monocot and dicot plant species.

The C14 fatty acids produced in the transgenic host cells of thisinvention are useful in various commercial applications, and will findparticular use, for example, in the detergent industry.

The following examples are provided by way of illustration and not byway of limitation.

EXAMPLES Example 1 Acyl-ACP Thioesterase Sequences

A. Cuphea hookeriana

DNA sequences corresponding to Cuphea thioesterase peptide regions areobtained by PCR using degenerate olgonucleotides designed from peptidefragments from conserved regions of plant thioesterases described in WO92/20236. A forward primer, TECU9, contains 17 nucleotides correspondingto all possible coding sequences for amino acids 176-181 of the bay andcamphor thioesterase proteins. A reverse primer, TECU3A, contains 18nucleotides corresponding to the complement of all possible codingsequences for amino acids 283-288 of the bay and camphor thioesteraseproteins, In addition, the forward and reverse primers contain BamHI orXhoI restriction sites, respectively, at the 5' end, and the reverseprimer contains an inosine nucleotide at the 3' end. The safflower, bayand camphor sequences diverge at two amino acid positions in the forwardprimer region, and at one amino acid residue in the reverse primerregion. The degeneracy of oligonucleotide primers is such that theycould encode the safflower, bay and camphor sequences.

Polymerase chain reaction samples (100 μl) are prepared using reversetranscribed Cuphea hookeriana RNA as template and 1 μM of each of theoligonucleotide primers. PCR products are analyzed by agarose gelelectrophoresis, and an approximately 300 bp DNA fragment, the predictedsize from the thioesterase peptide sequences, is observed. The DNAfragment, designated C93A (Cuphea) is isolated and cloned into aconvenient plasmid vector using the PCR-inserted BamHI and XhoIrestriction digest sites. DNA sequence of representative clones isobtained. Analysis of these sequences indicates that at least twodifferent, but homologous Cuphea hookeriana cDNAs were amplified.

Total Cuphea RNA for cDNA library construction may be isolated fromdeveloping Cuphea hookeriana embryos by modifying the DNA isolationmethod of Webb and Knapp (Plant Mol. Biol. Reporter (1990) 8:180-195).Buffers include:

REC: 50 mM TrisCl pH 9, 0.7M NaCl, 10 mM EDTA pH8, 0.5% CTAB.

REC+: Add B-mercaptoethanol to 1% immediately prior to use.

RECP: 50 mM TrisCl pH9, 10 mM EDTA pH8, and 0.5% CTAB.

RECP+: Add B-mercaptoethanol to 1% immediately prior to use.

For extraction of 1 g of tissue, 10 ml of REC+ and 0.5 g of PVPP isadded to tissue that has been ground in liquid nitrogen and homogenized.The homogenized material is centrifuged for 10 min at 1200 rpm. Thesupernatant is poured through miracloth onto 3 ml cold chloroform andhomogenized again. After centrifugation, 12,000 RPM for 10 min, theupper phase is taken and its volume determined. An equal volume of RECP+is added and the mixture is allowed to stand for 20 min. at roomtemperature. The material is centrifuged for 20 min. at 10,000 rpm twiceand the supernatant is discarded after each spin. The pellet isdissolved in 0.4 ml of 1M NaCl (DEPC) and extracted with an equal volumeof phenol/chloroform. Following ethanol preciptation, the pellet isdissolved in 1 ml of DEPC water. Poly (A) RNA may be isolated from thistotal RNA according to Maniatis et al. (Molecular Cloning: A LaboratoryManual (1982) Cold Springs Harbor, N.Y.). cDNA libraries may beconstructed in commercially available plasmid or phage vectors.

Thioesterase encoding fragments obtained by PCR as described above arelabeled and used to screen Cuphea cDNA libraries to isolate thioesterasecDNAs. Preliminary DNA sequence of a Cuphea cDNA clone TAA 342 ispresented in FIG. X. Translated amino acid sequence of the Cuphea clonefrom the presumed mature N-terminus (based on homology to the baythioesterase) is shown.

The sequence is preliminary and does not reveal a single open readingframe in the 5' region of the clone. An open reading frame believed torepresent the mature protein sequence is shown below the correspondingDNA sequence. The N-terminal amino acid was selected based on homologyto the bay thioesterase protein.

Additional Cuphea hookeriana cDNA clones were obtained by screening acDNA library prepared using a Uni-ZAP (Stratagene) phage library cloningsystem. The library was screening using radiolabeled TAA 342 DNA. Thelibrary was hybridized at 42° C. uing 30% formamide, and washing wasconducted at low stringency (room temperature with 1× SSC, 0.1% SDS).Numerous thioesterase clones were identified and DNA sequencesdetermined. Three classes of Cuphea cDNA clones have been identified.The original TAA 342 clone discussed above is representative of CUPH-1type clones which have extensive regions of homology to other plantmedium-chain preferring acyl-ACP thioesterases. Nucleic acid sequenceand translated amino acid sequence of a CUPH-1 clone, CMT9, is shown inFIG. 6 of WO 94/10288. The mature protein is believed to begin either ator near the leucine at amino acid position 88, or the leucine at aminoacid position 112. Northern analysis of RNA isolated from various Cupheahookeriana plant tissues indicates that the CUPH-1 gene is expressed ata low level in all Cuphea hookeriana plant tissues examined.

A second class of Cuphea thioesterase cDNAs is identified as CUPH-2.These cDNAs also demonstrate extensive homology to other plantmedium-chain acyl-ACP thioesterases. Expression of a representativeclone, CMT7, in E. coli demonstrated that CUPH-2 clones encode amedium-chain preferring acyl-ACP thioesterase protein havingpreferential activity towards C8 and C10 acyl-ACP substrates. DNAsequence and translated amino acid sequence of CMT7 is shown in FIG. 7of WO 94/10288.

Preliminary DNA sequence from the 5' end of an additional Cupheahookeriana clone, CMT13, is shown in FIG. 6 herein. Although CMT13demonstrates extensive sequence identity with CMT7, DNA sequencealignment reveals several gaps, which together total approximately 48nucleotides, where the CMT13 clone is missing sequences present in theCMT7 clone. CMT13 is also referred to as a CUPH-4 clone.

Northern analysis of RNA isolated from various Cuphea hookeriana planttissues indicates that CUPH-2 and CUPH-4 genes are highly expressed indeveloping seed tissues. Expression of the CUPH-2 and CUPH-4 clones inother C. hookeriana tissues, such as leaves, was not detected.

DNA sequence of an additional clone, CMT10, is shown in FIG. 9 of WO94/10288. CMT10 has greater than 90% sequence identity with CMT9, butless than the approximately 99% sequence identity noted in fragmentsfrom other CUPH-1 type clones. CMT10 is also referred to as a CUPH-5type clone.

B. Cuphea palustris

Total RNA is isolated from developing seeds of C. palustris as describedabove for C. hookeriana. A lambda ZipLox (BRL; Gaithersburg, Md.) cDNAlibrary containing approximately 6×10⁶ pfu is constructed from totalRNA. Approximately 500,000 plaques from the unamplified library arescreened using a mixed probe containing the thioesterase coding regionsfrom Cuphea hookeriana CUPH-1 (CMT-9), CUPH-2 (CMT-7) and CUPH-5(CMT-10). (DNA sequences of these clones are provided in WO 94/10288).Low stringency hybridization conditions are used: hybridization isconducted at room temperature in a solution of 30% formamide and 2× SSC(1× SSC=0.15M NaCl; 0.015M Na citrate). Eighty two putative positiveclones were identified, thirty of which were plaque purified.

The nucleic acid sequence and translated amino acid sequence of clonedesignated as MCT34 is provided in FIG. 1. The translated amino acidsequence of this clone is approximately 80% identical to the sequence ofa Cuphea hookeriana CUPH-4 clone (CMT-13 in FIG. 8 of WO 94/10288).

C. Bacterial luxD Gene Sequences

A Vibrio harvei luxD gene sequence is provided in Miyamoto et al. (J.Biol. Chem. (1988) 262:13393-13399). Additional luxD gene sequences havebeen reported by Baldwin et al. (J. Biolumin. Chemilum. (1989)4:326-341) and Cochrum et al. (Nucl. Acids Res. (1990) 18:5570).

D. Nutmeg (Myristica fragrans)

Total RNA is isolated from developing nutmeg seeds as described abovefor Cuphea species. A lambda Zap (Stratagene; La Jolla, Calif.) cDNAlibrary is constructed from total RNA. A BamHI/PstI fragment of pCGN3822containing approximately 900 bp of a bay thioesterase C12 preferringacyl-ACP thioesterase encoding sequence (FIG. 1 of WO 94/10288) isradiolabeled and used as a probe of the nutmeg cDNA library under thefollowing hybridization conditions: overnight hybridization at 30° C. in50% formamide, 2×SSC, 5% dextran sulfate. The hybridized filters arewashed at 30° C. in 0.1% SSC, 0.1%SDS and autoradiographed. Fiveputative positive clones were identified, three of which contain thesequence shown in FIG. 3, and are designated MYRF-2 or MfFatB1, and oneof which contained the sequence shown in FIG. 2, and which is designatedMYRF-1 or MfFatB2. Sequence of the other putative positive cloneindicated that it did not encode an acyl-ACP thioesterase.

Sequence analysis of the MYRF-1 and MYRF-2 clones indicates that MYRF-1is substantially a truncated version of MYRF-2, the initial prolineresidue of MYRF-1 corresponds to amino acid 97 of the MYRF-2 sequence.Another major difference in these clones is seen at the 3' end of thethioesterase encoding regions. The MYRF-1 clone lacks the TAG stop codonat nucleotides 1624-1626 of the MYRF-2 sequence, and thus the translatedamino acid sequence of MYRF-1 extends into the MYRF-2 3' untranslatedregion until the next available in frame stop codon is reached (TGA atnucleotides 1087-1089 of MYRF-1).

E. Camphor (Cinnamomum camphora)

DNA sequence and translated amino acid sequence of a Class II camphorthioesterase encoding region generated by PCR is provided in FIG. 5B ofWO 92/20236. A DNA fragment containing the mature protein region of thecamphor clone is obtained by PCR from reverse transcribed cDNA preparedusing RNA from developing camphor embryos. Forward (sense) and reverse(antisense) PCR primers, #4164 and #4165, are prepared which containsequences useful for cloning using the CLONEAMP™ system (GIBCO BRL;Gaithersburg, Md.). Oligonucleotide 4164 contains a 20 nucleotide regioncorresponding to the camphor thioesterase encoding sequence ofnucleotides 119-138 of the sequence in FIG. 5B of WO 92/20236.Oligonucleotide 4165 contains a 20 nucleotide region complementary tothe camphor thioesterase 3' untranslated sequence represented asnucleotides 1391-1410 of FIG. 5B in WO 92/20236. The sequences of 4164and 4165 are as follows: ##STR1## DNA sequence and translated amino acidsequence of a camphor PCR fragment obtained by PCR with 4164 and 4165are provided in FIG. 4. The sequence begins at the XbaI site located atthe beginning of the mature protein encoding region of the camphorthioesterase.

F. Elm

Elm acyl-ACP thioesterase clones may also be obtained using PCR primersfor plant thioesterase sequences as discussed above for Cuphea. TECU9and TECU3A are used in PCR reactions using reverse transcribed RNAisolated from elm embryos as template. As with Cuphea, an approximately300 nucleotide fragment, E93A, is obtained and used to probe an elm cDNAlibrary. Nucleic acid sequence and translated amino acid sequence of anelm medium-chain preferring acyl-ACP thioesterase clone, ULM-1, areshown in FIG. 5. The clone encodes the entire mature elm thioesteraseprotein, but appears to be lacking some of the transit peptide encodingregion. By comparison with other plant medium-chain acyl-ACPthioesterases, the mature elm protein is believed to begin either at theleucine indicated as amino acid number 54, or at the asparatateindicated as amino acid number 79.

Example 2 Expression of C14:0 Acyl-ACP Thioesterases in E. coli

A. Cuphea palustris

Constructs for expression of a Cuphea palustris acyl-ACP thioesteraseencoding sequence in E. coli are prepared. cDNA clone MCT34 is used astemplate for a polymerase chain reaction (PCR) to insert a StuI site 5'to the presumed mature protein start site located at amino acid 108 ofthe sequence shown in FIG. 1. A forward primer for PCR, MCT34 F1,contains DNA sequence corresponding to nucleotides 437-454 of the C.palustris sequence shown in FIG. 1, as well as sequences for insertionof SphI and StuI restriction digestion sites. An M13 sequencing primerreferred to as "M13 Forward" is used for priming the reverse, orantisense, reaction. Sequence of the PCR primers are as follows:##STR2##

The resulting PCR product is cloned as a StuI/XbaI fragment into pUC118,resulting in clone MCT34LZ, which provides for expression of the C.palustris thioesterase in E. coli as a lacZ fusion protein.

An additional construct for expression of the C. palustris thioesterasecDNA clone MCT34 in E. coli is prepared using a Qiagen (Chatsworth,Calif.) pQE vector which provides for high level expression and proteinpurification capability through a histidine tag. The DNA productresulting from PCR using the MCT34F1 and M13 Forward primers describedabove, is digested with SphI and SnaBI and cloned into SphI and SmaIdigested pQE30 (Qiagen), resulting in MCT34HT.

MCT34LZ is transformed into E. coli fadD, an E. coli mutant which lacksmedium-chain specific acyl-CoA synthetase (Overath et al., Eur. J.Biochem (1969) 7:559-574) for analysis of lipid composition. Cellscontaining the thioesterase construct, and a similar culture of controlcells are grown at 30° C. to an OD₆₀₀ of ˜0.5. Induction of thethioesterase expression may be achieved by the addition of IPTG to 0.2to 0.4 mM followed by further growth for 30 to 120 minutes. For slowgrowing cultures, longer growth periods may be required followingaddition of IPTG. A 4.5 ml sample of the E. coli cells is transferredinto a 15 ml glass vial with a teflon-lined cap. 100 μl of a 1 mg/mlstandards solution containing 1 mg/ml each of C11:0 free fatty acid,C15:0 free fatty acid, and C17:0 TAG in 1:1 chloroform/methanol is addedto the sample, followed by addition of 200 μl of glacial acetic acid and10 ml of 1:1 chloroform/methanol. The samples are vortexed to mixthoroughly and centrifuged for 5 minutes at 1000 rpm for complete phaseseparation. The lower (chloroform) phase is carefully removed andtransferred to a clean flask appropriate for use in a rotary evaporator(Rotovap). The sample is evaporated to near dryness. As medium-chainfatty acids appear to evaporate preferrentially after solvent isremoved, it is important to use just enough heat to maintain the vialsat room temperature and not completely remove the chloroform. The liquidresidue is measured and transferred to a 2 ml glass vial with a Tefloncap. The vial used in the rotary evaporator is washed withchloroform/methanol, and the chloroform/methanol sample is pooled withthe liquid residue (total volume of 600 μl).

For analysis of total fatty acids, a 100 μl aliquot of the sample ismethanolyzed by adding 1 ml of 5% sulfuric acid in methanol,transferring the samples to a 5 ml vial, and incubating the sample in a90° C. water bath for 2 hours. The sample is allowed to cool, afterwhich 1 ml of 0.9% NaCl and 300 μl of hexane are added. The sample isvortexed to mix thoroughly and centrifuged at 1000 rpm for 5 minutes.The top (hexane) layer is carefully removed and placed in a plasticautosampler vial with a glass cone insert, followed by capping of thevial with a crimp seal.

For analysis of free fatty acids, the following TLC procedure forseparation of free fatty acids from phospholipids (Cho and Cronan (1994)J. Bacterial. 1793-1795) is applied prior to methanolysis as describedabove. A 100 μl aliquot of the rotary evaporator residue and washsolution described above is applied to two lanes (50 μl/lane) of asilica-G TLC plate. The plates are developed in petroleumether/ether/acetic acid (70/30/2, v/v) for approximately 15-20 minutes.The phospholipids remain at the origin, while the neutral lipids migrateclose to the solvent front. Lipids are stained with iodine very briefly,marked and the silica from the marked areas transfered to Teflon-capped2 ml tubes. The respective areas from the two lanes are pooled, and thesamples are methanolyzed as described above.

Samples are analyzed by gas-liquid chromatography (GC) using atemperature program to enhance the separation of components having 10 orfewer carbons. The temperature program used provides for a temperatureof 140° C. for 3 minutes, followed by a temperature increase of 5°C./minute until 230° C. is reached, and 230° C. is maintained for 11minutes. Samples are analyzed on a Hewlett-Packard 5890 (Palo Alto,Calif.) gas chromatograph. Fatty acid content calculations are based onthe internal standards. Results are presented in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Free Fatty Acids (nmol/ml) in E. coli (fadD)                                  Strain   12:0    14:0    14:1  16:0  16:1  18:1                               ______________________________________                                        Control  1.87    0.54    0.0   1.70  0.0   0.0                                MCT34LZ  2.41    8.83    19    2.96  0.0   0.0                                ______________________________________                                    

The above results demonstrate a substantial increase in the productionof 14:0 and 14:1 fatty acids in cells transformed with the C. palustrisMCT34LZ clone.

B. C. hookeriana CUPH-4

A construct for expression of C. hookeriana CUPH-4 thioesterase in E.coli as a lacZ fusion is also prepared using PCR and cloning techniquessuch as described above for preparation of C. palustris constructs.

C. Nutmeg

Constructs for expression of two nutmeg (Myristica fragrans) Class IItype thioesterases, MYRF-1 (MfFatB2) and MYRF-2 (MfFatB1), in E. coli aslacZ fusion proteins are prepared. MfFatB1 and MfFatB2 are digested withSalI and XhoI to excise the clone fragments containing the thioesteraseencoding sequence from amino acid 131 of the MfFatB1 sequence (FIG. 3),or amino acid 35 of the MfFatB2 sequence (FIG. 2), through the 3' endsof the cDNA clones. The excised thioesterase encoding fragments areinserted into SalI digested pUC8 resulting in pCGN3856 (MfFatB1) andpCGN3855 (MfFatB2). These constructs encode lacZ fusions of theapproximate mature thioesterase protein sequence (amino acid 130 of theMfFatB1 preprotein was selected as the mature protein N-terminus byhomology to bay thioesterase protein).

The fusion proteins are expressed in fad+ and fadD strains of E. coliK12. Analysis of total fatty acids in liquid cultures of MYRF-1 andMYRF-2 transformed K27 (fadD) after overnight growth at 30° C. areprovided in Table 2 below.

D. Camphor

The camphor PCR fragment described above is cloned into a pAMP vectorresulting in pCGN5219. pCGN5219 is digested with XbaI and SalI and theresulting camphor thioesterase fragemnt is cloned into XbaI and SalIdigested pBCSK+ (Stratagene), resulting in pCGN5220. pCGN5220 is used totransform E. coli fadD for analysis of lipid composition as describedabove. Results of these analyses are provided in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        Total Fatty Acids (nmol/ml) in E. coli (fadD)                                 Strain   12:0    14:0    14:1  16:0  16:1  18:1                               ______________________________________                                        Control  3       19      2     141   59    42                                 MYRF-1   19      277     19    121   299   54                                 MYRF-2   32      240     31    47    296   17                                 CINC-1   99      195     204   43    102   26                                 CUPH-4   3       217     0     277   107   112                                ______________________________________                                    

In comparison to the control, 14:0 and 16:1 fatty acids are drasticallyelevated for the nutmeg, camphor and C. hookeriana clones. Increases in12:0 and 14:1 are also observed with the nutmeg and camphor clones, andincreases in 16:0 and 18:1 are also seen with the C. hookeriana CUPH-4clone.

E. Elm

An elm acyl-ACP thioesterase cDNA clone is expressed in E. coli as alacZ fusion. The ULM1 cDNA clone, KA10, represented in FIG. 5 isdigested with StuI and XbaI to produce an approximately 1000 base pairfragment containing the majority of the mature elm thioesterase encodingsequence. The StuI site is located at nucleotides 250-255 of thesequence shown in FIG. 5, and the XbaI site is located at nucleotides1251-1256, 3' to the stop codon. As discussed above, the N-terminus forthe mature elm thioesterase is believed to be either the leucine residueencoded by nucleotides 160-162 or the aspartate residue encoded bynucleotides 235-237. The StuI/XbaI fragment is inserted into StuI/XbaIdigested pUC118 resulting in construct KA11. For expression analysis,KA11 is used to transform E. coli strain DH5å or fadD.

As has been observed with bay thioesterase expression constructs WO92/20236), E. coli clones expressing elm thioesterase exhibited abnormalgrowth rate and morphology phenotypes. The growth rate of E. coli DH5å(fadD₊) or fadD mutant cells expressing the elm thioesterase isinitially much slower than growth of control cells at either 25° C. or30° C. At 37° C., the elm thioesterase plasmid appears to be toxic tothe E. coli cells. After growing the transformed cultures for severalgenerations, variants may be selected which grow at the same rate ascontrol cells at 25° C. or 30° C. A similar result was seen with fadDcells comprising bay thioesterase expression constructs. A fadD mutantstrain selected as having a normal growth rate when expressing the baythioesterase was cured of the bay thioesterase construct and transformedwith the elm thioesterase construct. This strain exhibits a normalgrowth phenotype in the first generation of cells comprising the elmthioesterase construct.

The activity assays from normal growth phenotype KA11 cells reproduciblydemonstrate differentially elevated C10:0-ACP and C16:0-ACP hydrolysisactivities. Upon induction with IPTG, the C10:0-ACP and C16:-ACPactivities are affected differently. The specific activity of theC16:0-ACP hydrolysis decreases slightly, while that of the C10:0-ACPhydrolase increases by approximately 44%. This data suggests thepossibility that C16:0-ACP hydrolysis activity is derived from the E.coli cells, rather than the elm thioesterase. However, lipid analysis oftransgenic plant seeds expressing the elm thioesterase (Example 5)demonstrates increased production of C16:0 resulting from C16:0-ACPhydrolysis activity of the elm thioesterase.

Results of GC analysis of total fatty acids in overnight culturestransformed with elm thioesterase construct KA11 are provided in Table 3below.

                  TABLE 3                                                         ______________________________________                                        Total Fatty Acids (nmol/ml) in E. coli (fadD)                                 Strain 8:0    10:0   12:0  14:0 14:1 16:0 16:1  18:1                          ______________________________________                                        Control                                                                               5      7      3     21   2   127  44    27                            KA11   96     161    32    109  16    17  91    17                            ______________________________________                                    

Substantial increases in C8, C10, C12 and C14 fatty acids were observed.Subsequence studies of free fatty acids in overnight cultures of KA11demosntrate that the observed increases are due to the free fatty acidcompositions. In addition, an increase in 16:0 free fatty acids in KA11versus the control culture was also observed.

F. Assay for Thioesterase Activity

For thioesterase activity assays, E. coli cells containing the acyl-ACPthioesterase constructs, and a similar culture of control cells aregrown at 30° C. to an OD₆₀₀ of ˜0.5. Induction of thioesteraseexpression in lacZ fusion constructs may be achieved by the addition ofIPTG to 0.4 mM followed by 1 or 2 hours further growth. For slow growingcultures, longer growth periods may be required following addition ofIPTG.

A ten-ml aliquot of each culture (containing cells plus the culturemedium) is assayed for specific activity towards various carbon chainlength acyl-ACP substrates as follows. Cells are harvested bycentrifugation, resuspended in 0.5 ml assay buffer and lysed bysonication. Cell debris may be removed by further centrifugation. Thesupernatant is then used in thioesterase activity assays as per Pollardet al., Arch. Biochem & Biophys. (1991) 281:306-312. Results ofthioesterase activity assays on Cuphea, nutmeg, elm and camphorthioesterase clones using 8:0, 10:0, 12:0, 14:0, 16:0, 18:0 and 18:1acyl-ACP substrates are provided in Table 4 below. Results are presentedas relative activity of the thioesterase expressing cells compared tocontrol cells.

                  TABLE 4                                                         ______________________________________                                        Relative Activity (TE/Control)                                                Strain   8:0    10:0    12:0 14:0  16:0 18:0  18:1                            ______________________________________                                        MCT34HT  0.9    0.8     1.0  42.8  21.8 1.5                                   MYRF-1   1.1    1.4     1.8  13.6  13.3 5.5   13.6                            MYRF-2   0.9    0.9     0.8  4.2   6.6  2.8   10.9                            CINC-1          1.3     1.9  8.9   2.0  1.1   1.1                             ULM-1           5.4     1.5        6.9        1.7                             ______________________________________                                    

Substantial increases in the hydrolysis activity on 14:0 and 16:0relative to the control cells are observed with C. palustris MCT34HTtransformed cells. Cells transformed with the nutmeg MYRF-1 and MYRF-2clones also demonstrate substantial increases in activity on 14:0 and16:0 substrates, as well as less substantial increases with 18:0 and18:1. Expression of the camphor CINC-1 clone results mainly in increasedactivity on 14:0, although a lesser increase in 16:0 hydrolysis activityis also observed.

G. Bacterial luxD Gene

A Vibrio harvei myristoyl ACP thioesterase (luxD) encoding sequence(Miyamoto et al., J. Biol. Chem. (1988) 262:13393-13399) lacking theinitial ATG codon is prepared by PCR. The gene is expressed in E. colias a lacZ fusion and E. coli extracts are assayed to confirm myristoylACP thioesterase activity. The C14 thioesterase construct is used totransform an E. coli fadD strain. The cells transformed in this mannerdeposit large quantities of crystals which are identified as potasssiummyristate by mass spectrometry. Fatty acid analysis of the E. coliextracts reveals that greater than 50% (on a mole basis) of the fattyacids are C14:0, as compared to control E. coli fadD cells which containapproximately 11.5 mole percent C14:0.

Example 3 Constructs for Plant Transformation

A. Napin Expression Cassette

A napin expression cassette, pCGN1808, is described in copending U.S.patent application Ser. No. 07/742,834 which is incorporated herein byreference. pCGN1808 is modified to contain flanking restriction sites toallow movement of only the expression sequences and not the antibioticresistance marker to binary vectors. Synthetic oligonucleotidescontaining KpnI, NotI and HindIII restriction sites are annealed andligated at the unique HindIII site of pCGN1808, such that only oneHindIII site is recovered. The resulting plasmid, pCGN3200 containsunique HindIII, NotI and KpnI restriction sites at the 3'-end of thenapin 3'-regulatory sequences as confirmed by sequence analysis.

The majority of the napin expression cassette is subcloned from pCGN3200by digestion with HindIII and SacI and ligation to HindIII and SacIdigested pIC19R (Marsh, et al. (1984) Gene 32:481-485) to make pCGN3212.The extreme 5'-sequences of the napin promoter region are reconstructedby PCR using pCGN3200 as a template and two primers flanking the SacIsite and the junction of the napin 5'-promoter and the pUC backbone ofpCGN3200 from the pCGN1808 construct. The forward primer contains ClaI,HindIII, NotI, and KpnI restriction sites as well as nucleotides 408-423of the napin 5'-sequence (from the EcoRV site) and the reverse primercontains the complement to napin sequences 718-739 which include theunique SacI site in the 5'-promoter. The PCR was performed using in aPerkin Elmer/Cetus thermocycler according to manufacturer'sspecifications. The PCR fragment is subcloned as a blunt-ended fragmentinto pUC8 (Vieira and Messing (1982) Gene 19:259-268) digested withHincII to give pCGN3217. Sequenced of pCGN3217 across the napin insertverifies that no improper nucleotides were introduced by PCR. The napin5-sequences in pCGN3217 are ligated to the remainder of the napinexpression cassette by digestion with ClaI and SacI and ligation topCGN3212 digested with ClaI and SacI. The resulting expression cassettepCGN3221, is digested with HindIII and the napin expression sequencesare gel purified away and ligated to pIC20H (Marsh, supra) digested withHindIII. The final expression cassette is pCGN3223, which contains in anampicillin resistant background, essentially identical 1.725 napin 5'and 1.265 3' regulatory sequences as found in pCGN1808. The regulatoryregions are flanked with HindIII, NotI and KpnI restriction sites andunique SalI, BglII, PstI, and XboI cloning sites are located between the5' and 3' noncoding regions.

B. Oleosin Expression Cassette

A cassette for cloning of sequences for transcription under theregulation of 5' and 3' regions from an oleosin gene may be prepared.Sequence of a Brassica napus oleosin gene is provided by Lee and Huang(Plant Phys. (1991) 96:1395-1397). Primers to the published sequence areused in PCR reactions to obtain the 5' and 3' regulatory regions of anoleosin gene from Brassica napus cv. Westar. Two PCR reactions wereperformed, one to amplify approximately 950 nucleotides immediatelyupstream of the ATG start codon for the oleosin gene, and one to PCRamplify approximately 600 bp including and downstream of the TAA stopcodon for the oleosin gene. The PCR products were cloned into plasmidvector pAMP1 (BRL) according to manufacturer's protocols to yieldplasmids pCGN7629 which contains the oleosin 5' flanking region andpCGN7630 which contains the 3' flanking region. The PCR primers includedconvenient restriction sites for cloning the 5' and 3' flanking regionstogether into an expression cassette. A PstI fragment containing the 5'flanking region from pCGN7629 was cloned into PstI digested pCGN7630 toyield plasmid pCGN7634. The BssHII (New England BioLabs) fragment frompCGN7634, which contains the entire oleosin expression cassette wascloned into BssHII digested pBCSK+ (Stratagene) to provide the oleosincassette in a plasmid, pCGN7636. Sequence of the oleosin cassette inpCGN7636 is provided in FIG. 7. The oleosin cassette is flanked byBssHII, KpnI and XbaI restriction sites, and contains SalI, BamHI andPstI sites for insertion of wax synthase, reductase, or other DNAsequences of interest between the 5' and 3' oleosin regions.

C. C. palustris Acyl-ACP Thioesterase Expression Constructs

Constructs for expression of C. palustris thioesterase cDNA clone MCT34in plant seeds under the regulatory control of napin and oleosinregulatory regions are prepared as follows. The thioesterase encodingregion from MCT34 is obtained by PCR amplification usingoligonucleotides for insertion of a SalI site 5' to the ATG start codon,and an NsiI site immediately 3' to the MCT34 translation stop codon. Theoligonucleotide primers for PCR contained the SalI site (CpMet-1 forwardprimer) and the NsiI site (CpStop-1 reverse primer). In addition, theprimers contain "CAU" (forward primer) and "CUA" (reverse primer) repeatsequences for cloning using the CLONEAMP™ system. Sequence of the PCRprimers is as follows: ##STR3## The resulting PCR product is cloned intopAMP and the DNA sequence determined to verify the PCR products.

The C. palustris thioesterase pAMP clone (pCGN3860) is digested withSalI and NsiI and the thioesterase encoding fragment isolated and clonedinto SalI/PstI digested pCGN3223 (napin expression cassette) or pCGN7636(oleosin expression cassette), resulting in pCGN3861 and pCGN3862,respectively.

Binary vectors for plant transformation with the C. palustris expressionconstructs are prepared by digestion of pCGN3861 and pCGN3862 withAsp718 and insertion of the resulting fragments into Asp718 digestedpCGN1578 (McBride et al. (1990) Plant Mol. Biol. 14:269-276), resultingin pCGN3863 and pCGN3864, respectively.

D. luxD Expression Construct

Constructs for expression of the Vibrio harvei myristoyl ACPthioesterase in plant cells which utilize napin promoter regions areprepared as follows. Two 100 base oligos are synthesized: ##STR4##

The two oligos contain a region of complementary sequence for annealing(underlined region). A TAQ polymerase extension reaction utilizing thetwo oligos yields a 180 bp product. The oligos consisted essentially ofluxD sequence with sequence changes introduced to remove the 3 potentialpoly(A) addition sites and to alter 5 bases to change the codonpreference from bacteria to plants. All changes were conservative; i.e.the amino acid sequence was not altered.

The 180 bp TAQ polymerase extension product is blunted and cloned intoBluescript. The approximately 180 bp luxD fragment is then removed fromBluescript by digestion with XbaI and EaeI and cloned in frame with theEaeI/XbaI fragment from the Vibrio cDNA clone, containing the remainderof the luxD gene, by 3-way ligation into XbaI/XhoI digested BluescriptSK. The luxD gene is removed by digestion with XbaI and partialdigestion with PstI and cloned in frame with the safflower thioesterasetransit peptide encoding region into a napin expression casette. Thenapin 5'/safflower transit:myristoyl ACP thioesterase/napin 3' fragmentis cloned into KpnI/BamHI digested pCGN1557 (McBride and Summerfelt,supra) resulting in pCGN3845, a binary expression vector for planttransformation.

E. Nutmeg Acyl-ACP Thioesterase Expression Construct

Constructs for expression of nutmeg thioesterase cDNA clone MfFatB1(pCGN3856 or MYRF-2) in plant seeds under the regulatory control ofnapin and oleosin regulatory regions are prepared as follows. Thethioesterase encoding region from MfFatB1 is obtained by PCRamplification using oligonucleotides for insertion of a BamHI site 5' tothe ATG start codon, and an XhoI site 3' to the MfFatB1 translation stopcodon. The oligonucleotide primers for PCR contained the BamHI site(forward or sense primer) and the XhoI site (reverse or antisenseprimer). In addition, the primers contain "CAU" (forward primer) and"CUA" (reverse primer) repeat sequences for cloning using the CLONEAMP™system.

Sequence of the PCR primers is as follows: ##STR5## The resulting PCRproduct is cloned into pAMP and the DNA sequence determined to verifythe PCR products.

The nutmeg thioesterase pAMP clone (TA431) is digested with XhoI andpartially digested with BamHI. The thioesterase encoding fragment isisolated (1.3 kb band) and cloned into BglII/Xho digested pCGN3223(napin expression cassette), resulting in pCGN3868. A binary vector forplant transformation with the nutmeg expression construct is prepared bydigestion of pCGN3868 with Asp718, and insertion of the resulting napin5'/nutmeg TE/napin 3' fragment (4.2 kb) into pCGN1578PASS at the Asp718site. pCGN1578PASS is prepared from pCGN1578 (McBride et al., supra) bysubstitution of the pCGN1578 polylinker region with a polylinker regioncontaining the following restriction sites: Asp718, Asc, Pac, Swa, Sseand HindIII.! The resulting construct, pCGN3854, is used for planttransformation for production of C14 fatty acids.

A construct for expression of the nutmeg thioesterase under theregulatory control of an oleosin promoter is prepared as follows.pCGN3868 (napin 5'/nutmeg TE/napin 3' expression construct describedabove) is digested with SalI and EcoRV, and the resulting fragment,containing the nutmeg thioesterase encoding region joined in the 5' to3' orientation to the napin 3' regulatory region, is inserted into SalIand EcoRV digested pCGN7636 (oleosin expression cassette describedabove). The resulting construct, pCGN3858, contains an oleosin 5'/nutmegTE/napin 3'/oleosin 3' construct. pCGN3858 is digested with Asp718 andpartially digested with BamHI to produce an ˜2.6 kb fragment containingthe oleosin 5', nutmeg thioesterase encoding region, and ˜320nucleotides of the napin 3' regulatory region. The 2.6 kb fragment iscloned into Asp718/BamHI digested pCGN1578, resulting in pCGN3857, abinary vector for plant transformation and expression of the nutmegthioesterase.

F. Camphor Acyl-ACP Thioesterase Expression Construct

A construct for expression of camphor thioesterase under the regulatorycontrol of a napin promoter is described. A transit peptide encodingsequence for bay thioesterase is obtained by digestion of pCGN3826 (bayC12 preferring acyl-ACP thioesterase clone described in WO 92/20236)with XbaI and SalI generating a DNA fragment having a plasmid vectorbackbone and the bay transit peptide encoding sequence (XbaI site is atbeginning of mature bay protein encoding region). pCGN5220 (Example 2D)is digested with XbaI and Sal' to obtain the camphor mature TE encodingregion. The pCGN5220 and pCGN3826 SalI/XbaI fragments are ligated toproduce pCGN5231. pCGN5231 is digested with BamHI and SalI, and theresulting bay transit::camphor mature encoding fragment is inserted intoBglII/XhoI digested pCGN3223 (napin expression cassette), resulting inpCGN5232. pCGN5232 was digested with NotI and, with Klenow to produceblunt ends, and the resulting napin 5'/bay transit::camphor mature/napin3' fragment is inserted into HindIII digested and Klenow-bluntedpCGN1578. The resulting construct, pCGN5233, is a binary vector forplant transformation and expression of camphor thioesterase.

G. Elm Acyl-ACP Thioesterase Expression Construct

A construct for expression of an elm acyl-ACP thioesterase in plant seedcells using a napin expression cassette is prepared as follows. The elmULM-1 medium-chain acyl-ACP thioesterase cDNA does not appear to encodethe entire thioesterase transit peptide. Thus, the elm thioesterasecoding region was fused to the transit peptide encoding region from theCuphea CUPH-1 clone as follows. pCGN4800 (CUPH-1 in napin cassette) wasdigested with XbaI, blunted and digested with StuI to remove the matureprotein coding portion of the CUPH-1 construct. The StuI site is locatedat nucleotides 496-501 of the CUPH-1 sequence shown in FIG. 5 of WO94/10288. The XbaI site is located between the end of the Cupheathioesterase cDNA sequence and the napin 3' regulatory region. The ULM-1mature protein encoding region is inserted into the napin/Cuphea transitpeptide backbone resulting from removal of the Cuphea mature proteinendoding region as follows. The ULM-1 clone is digested with XbaI,blunted and digested with StuI to obtain the elm thioesterase matureprotein encoding region. The StuI site is located at nucleotides 250-255of the sequence shown in FIG. 2 of WO 94/10288, and the XbaI site islocated at nucleotides 1251-1256, 3' to the stop codon. Ligation of theelm StuI/XbaI fragment into the napin/Cuphea transit peptide backboneresults in pCGN4802, having the napin 5'/Cuphea transit:elm mature/napin3' expression construct. pCGN4802 is transferred to pCGN1557 as aHindIII fragment resulting in pCGN4803, a binary construct for planttransformation.

Example 4 Plant Transformation

A. Brassica Transformation

Brassica species may be transformed as reported by Radke et al. (PlantCell Reports (1992) 11:499-505; Theor. Appl. Genet. (1988) 75:685-694),or as described in detail below.

Brassica napus seeds are soaked in 95% ethanol for 2 min. surfacesterilized in a 1.0% solution of sodium hypochlorite containing a dropof Tween 20 for 45 min., and rinsed three times in sterile, distilledwater. Seeds are then plated in Magenta boxes with 1/10th concentrationof Murashige minimal organics medium (Gibco; Grand Island, N.Y.)supplemented with pyriodoxine (50 μg/l), nicotinic acid (50 μg/l),glycine (200 μ/l), and 0.6% Phytagar (Gibco) pH 5.8. Seeds aregerminated in a Percival chamber at 22° C. in a 16 h photoperiod withcool fluorescent and red light of intensity approximately 65 μgEinsteins per square meter per second (μEm⁻² S⁻¹).

Hypocotyls are excised from 5-7 day old seedlings, cut into piecesapproximately 4 mm in length, and plated on feeder plates (Horsch etal., Science (1985) 227:1229-1231). Feeder plates are prepared one daybefore use by plating 1.0 ml of a tobacco suspension culture onto apetri plate (100×25 mm) containing about 30 ml MS salt base (CarolinaBiological, Burlington, N.C.) 100 mg/l inositol, 1.3 mg/l thiamine-HCl,200 mg KH₂ PO₄ with 3% sucrose, 2,4-D (1.0 mg/l), 0.6% w/v Phytagar, andpH adjusted to 5.8 prior to autoclaving (MS 0/1/0 medium). A sterilefilter paper disc (Whatman 3 mm) is placed on top of the feeder layerprior to use. Tobacco suspension cultures are subcultured weekly bytransfer of 10 ml of culture into 100 ml fresh MS medium as describedfor the feeder plates with 2,4-D (0.2 mg/l), Kinetin (0.1 mg/l). Inexperiments where feeder cells are not used hypocotyl explants are cutand placed onto a filter paper disc on top of MS0/1/0 medium. Allhypocotyl explants are preincubated on feeder plates for 24 h. at 22° C.in continuous light of intensity 30 μEm⁻² S⁻¹ to 65 μEM⁻² S⁻¹.

Single colonies of A. tumefaciens strain EHA 101 containing a binaryplasmid are transferred to 5 ml MG/L broth and grown overnight at 30° C.Hypocotyl explants are immersed in 7-12 ml MG/L broth with bacteriadiluted to 1×10⁸ bacteria/ml and after 10-25 min. are placed onto feederplates. Per liter MG/L broth contains 5 g mannitol, 1 g L-Glutamic acidor 1.15 g sodium glutamate, 0.25 g kH₂ PO₄, 0.10 g NaCl, 0.10 gMGSO₄.7H₂ O, 1 mg biotin, 5 g tryptone, and 2.5 g yeast extract, and thebroth is adjusted to pH 7.0. After 48 hours of co-incubation withAgrobacterium, the hypocotyl explants are transferred to B5 0/1/0 callusinduction medium which contains filter sterilized carbenicillin (500mg/l, added after autoclaving) and kanamycin sulfate (BoehringerMannheim; Indianapolis, Ind.) at concentrations of 25 mg/l.

After 3-7 days in culture at 65 μEM⁻² S⁻¹ continuous light, callustissue is visible on the cut surface and the hypocotyl explants aretransferred to shoot induction medium, B5BZ (B5 salts and vitaminssupplemented with 3 mg/l benzylaminopurine, 1 mg/l zeatin, 1% sucrose,0.6% Phytagar and pH adjusted to 5.8). This medium also containscarbenicillin (500 mg/l) and kanamycin sulfate (25 mg/l). Hypocotylexplants are subcultured onto fresh shoot induction medium every twoweeks.

Shoots regenerate from the hypocotyl calli after one to three months.Green shoots at least 1 cm tall are excised from the calli and placed onmedium containing B5 salts and vitamins, 1% sucrose, carbenicillin (300mg/l), kanamycin sulfate (50 mg/l) and 0.6% w/v Phytagar). After 2-4weeks shoots which remain green are cut at the base and transferred toMagenta boxes containing root induction medium (B5 salts and vitamins,1% sucrose, 2 mg/l indolebutyric acid, 50 mg/l kanamycin sulfate and0.6% Phytagar). Green rooted shoots are tested for thioesteraseactivity.

B. Arabidposis Transformation

Transgenic Arabidopsis thaliana plants may be obtained byAgrobacterium-mediated transformation as described by Valverkens et al.,(Proc. Nat. Acad. Sci. (1988) 85:5536-5540). Constructs are transformedinto Agrobacterium cells, such as of strain EHA101 (Hood et al., J.Bacteriol (1986) 168:1291-1301), by the method of Holsters et al. (Mol.Gen. Genet. (1978) 163:181-187).

C. Peanut Transformation

DNA sequences of interest may be introduced as expression cassettes,comprising at least a promoter region, a gene of interest, and atermination region, into a plant genome via particle bombardment asdescribed in European Patent Application 332 855 and in co-pendingapplication U.S. Ser. No. 07/225,332, filed Jul. 27, 1988.

Briefly, tungsten or gold particles of a size ranging from 0.5 μM-3 μMare coated with DNA of an expression cassette. This DNA may be in theform of an aqueous mixture or a dry DNA/particle precipitate.

Tissue used as the target for bombardment may be from cotyledonaryexplants, shoot meristems, immature leaflets, or anthers.

The bombardment of the tissue with the DNA-coated particles is carriedout using a Biolistics™ particle gun (Dupont; Wilmington, Del.). Theparticles are placed in the barrel at variable distances ranging from 1cm-14 cm from the barrel mouth. The tissue to be bombarded is placedbeneath the stopping plate; testing is performed on the tissue atdistances up to 20 cm. At the moment of discharge, the tissue isprotected by a nylon net or a combination of nylon nets with meshranging from 10 μM to 300 μM.

Following bombardment, plants may be regenerated following the method ofAtreya, et al., (Plant Science Letters (1984) 34:379-383). Briefly,embryo axis tissue or cotyledon segments are placed on MS medium(Murashige and Skoog, Physio. Plant. (1962) 15:473) (MS plus 2.0 mg/l6-benzyladenine (BA) for the cotyledon segments) and incubated in thedark for 1 week at 25°±2° C. and are subsequently transferred tocontinuous cool white fluorescent light (6.8 W/m²). On the 10th day ofculture, the plantlets are transferred to pots containing sterile soil,are kept in the shade for 3-5 days are and finally moved to greenhouse.

The putative transgenic shoots are rooted. Integration of exogenous DNAinto the plant genome may be confirmed by various methods know to thoseskilled in the art.

Example 5 Analysis of Transgenic Plants

A. V. harveyi luxD Expression Construct

Transgenic plants comprising a construct for expression of the V.harveyi luxD gene under the regulatory control of a napin promoter(pCGN3845) are grown to seed and analyzed to determine the percentage ofC14 fatty acids produced as the result of insertion of the bacterialacyl transferase gene. Analysis of pooled seed samples from 24segregating transgenic (T1) Brassica napus plants indicates C14 fattyacid levels ranging from 0.12 to 1.13 mole %. Two plants, 3845-1 and3845-18, contain greater than 1 mole % C14:0 fatty acids in their seedoils. Similar analysis of non-transgenic B. napus seeds reveals C14:0levels of approximately 0.1 mole %. Analysis of single seeds from3845-18 reveals individual seeds having greater than 2 mole % C14:0 inthe oil. Western analysis is conducted to determine amounts of the C14:0thioesterase present in transgenic plants. A comparison of proteinamount to mole % C14:0 (myristate) produced indicates that myristatelevels increase with increasing amounts of the thioesterase protein.

B. Nutmeg (MYRF-2) Expression Construct

Mature seeds were harvested from transgenic Brassica napus plants (aQL01 derived low linolenic variety) containing pCGN3854, a construct forexpression of nutmeg thioesterase clone MYRF-2 under the regulatorycontrol of a napin promoter, and analyzed to determine mole percentfatty acid composition. Results are presented in Table 5 below.

                                      TABLE 5                                     __________________________________________________________________________    Plant                                                                             8:0                                                                              10:0                                                                              12:0                                                                             14:0                                                                              16:0                                                                             16:1                                                                              18:0                                                                             18:1                                                                              18:2                                                                             18:3                                       __________________________________________________________________________    3854-1                                                                            0.00                                                                             0.26                                                                              0.30                                                                             13.50                                                                             22.10                                                                            0.49                                                                              4.84                                                                             37.62                                                                             16.26                                                                            1.96                                       3854-2                                                                            0.00                                                                             0.42                                                                              0.26                                                                             14.91                                                                             29.05                                                                            0.52                                                                              6.87                                                                             25.76                                                                             17.73                                                                            1.70                                       3854-3                                                                            0.00                                                                             0.27                                                                              0.43                                                                             21.73                                                                             30.90                                                                            0.43                                                                              6.48                                                                             19.54                                                                             15.85                                                                            1.62                                       3854-4                                                                            0.00                                                                             0.33                                                                              0.28                                                                             15.24                                                                             26.89                                                                            0.51                                                                              6.28                                                                             29.61                                                                             16.33                                                                            1.84                                       __________________________________________________________________________

C14 fatty acyl groups are present in all four transgenic plantsanalyzed, with levels of C14 ranging from 13.5 to 21.73 mole percent.Background levels of C14 in non-transformed control plants areapproximately 0.1 mole percent. Single seeds from transformant 3854-3are disected for half seed lipid analysis. Results from these analysesare presented in Table 6 below.

                                      TABLE 6                                     __________________________________________________________________________    NO. 8:0                                                                              10:0                                                                              12:0                                                                             14:0                                                                              16:0                                                                             16:1                                                                              18:0                                                                             18:1                                                                              18:2                                                                             18:3                                       __________________________________________________________________________    1   0.00                                                                             2.31                                                                              0.40                                                                             19.11                                                                             29.78                                                                            0.36                                                                              6.63                                                                             22.69                                                                             14.08                                                                            1.46                                       2   0.00                                                                             1.84                                                                              0.37                                                                             20.26                                                                             30.45                                                                            0.44                                                                              5.76                                                                             19.30                                                                             17.49                                                                            1.51                                       3   0.00                                                                             1.53                                                                              0.37                                                                             16.87                                                                             29.24                                                                            0.49                                                                              8.11                                                                             25.66                                                                             13.06                                                                            1.44                                       4   0.00                                                                             2.39                                                                              0.44                                                                             20.82                                                                             29.96                                                                            0.58                                                                              6.02                                                                             18.47                                                                             16.78                                                                            1.68                                       5   0.00                                                                             2.91                                                                              0.43                                                                             19.66                                                                             30.16                                                                            0.62                                                                              6.71                                                                             23.12                                                                             12.43                                                                            1.48                                       6   0.00                                                                             2.01                                                                              0.40                                                                             18.52                                                                             29.50                                                                            0.36                                                                              6.99                                                                             23.49                                                                             14.01                                                                            1.67                                       7   0.00                                                                             4.02                                                                              0.51                                                                             23.04                                                                             29.94                                                                            0.25                                                                              5.30                                                                             17.16                                                                             16.13                                                                            1.75                                       8   0.00                                                                             3.13                                                                              0.38                                                                             18.03                                                                             27.87                                                                            0.58                                                                              6.09                                                                             25.51                                                                             14.31                                                                            1.51                                       9   0.00                                                                             3.00                                                                              0.44                                                                             21.23                                                                             29.19                                                                            0.63                                                                              5.68                                                                             17.58                                                                             18.34                                                                            1.85                                       12  0.00                                                                             2.52                                                                              0.34                                                                             17.80                                                                             28.93                                                                            0.38                                                                              6.73                                                                             23.62                                                                             15.11                                                                            1.53                                       13  0.00                                                                             2.56                                                                              0.45                                                                             21.48                                                                             30.59                                                                            0.49                                                                              6.02                                                                             18.43                                                                             16.02                                                                            1.48                                       14  0.00                                                                             2.19                                                                              0.39                                                                             18.40                                                                             30.48                                                                            0.47                                                                              7.44                                                                             23.39                                                                             12.68                                                                            1.42                                       15  0.00                                                                             1.88                                                                              0.28                                                                             15.17                                                                             28.81                                                                            0.44                                                                              7.73                                                                             28.03                                                                             12.94                                                                            1.35                                       16  0.00                                                                             2.10                                                                              0.38                                                                             19.83                                                                             30.34                                                                            0.43                                                                              6.23                                                                             20.33                                                                             15.95                                                                            1.33                                       17  0.00                                                                             2.44                                                                              0.42                                                                             18.73                                                                             28.89                                                                            0.60                                                                              7.21                                                                             22.26                                                                             14.83                                                                            1.68                                       18  0.00                                                                             2.77                                                                              0.45                                                                             20.32                                                                             29.55                                                                            0.47                                                                              6.55                                                                             21.93                                                                             13.93                                                                            1.46                                       19  0.00                                                                             3.37                                                                              0.40                                                                             17.72                                                                             27.95                                                                            0.48                                                                              6.38                                                                             24.22                                                                             14.01                                                                            2.01                                       20  0.00                                                                             2.40                                                                              0.36                                                                             19.72                                                                             29.92                                                                            0.50                                                                              6.72                                                                             19.79                                                                             16.10                                                                            1.52                                       __________________________________________________________________________

Additional single seed fatty acid composition data from 3854-3 and854-11 are presented in FIG. 8. These data indicate C14 levels of up to23% are obtained by expression of nutmeg thioesterase.

C. Camphor Expression Construct

Mature seeds were harvested from transgenic Brassica napus plantscontaining pCGN5233, a construct for expression of camphor thioesteraseclone CINC-1 under the regulatory control of a napin promoter, andanalyzed to determine mole percent fatty acid composition. Results arepresented in Table 7 below.

                                      TABLE 7                                     __________________________________________________________________________    Plant                                                                             8:0                                                                              10:0                                                                              12:0                                                                             14:0                                                                              16:0                                                                             16:1                                                                              18:0                                                                             18:1                                                                              18:2                                                                             18:3                                       __________________________________________________________________________    5233-1                                                                            0.00                                                                             0.84                                                                              0.87                                                                             6.93                                                                              9.64                                                                             0.95                                                                              1.51                                                                             41.35                                                                             21.47                                                                            14.99                                      5233-2                                                                            0.00                                                                             0.85                                                                              0.38                                                                             3.83                                                                              8.41                                                                             0.70                                                                              1.43                                                                             46.33                                                                             21.71                                                                            14.98                                      5233-4                                                                            0.00                                                                             0.96                                                                              1.33                                                                             11.46                                                                             11.69                                                                            1.06                                                                              1.16                                                                             31.07                                                                             23.54                                                                            16.26                                      5233-5                                                                            0.00                                                                             0.69                                                                              0.93                                                                             8.77                                                                              10.38                                                                            0.90                                                                              1.48                                                                             42.22                                                                             19.88                                                                            13.41                                      5233-6                                                                            0.00                                                                             0.69                                                                              1.29                                                                             11.38                                                                             10.98                                                                            0.83                                                                              1.54                                                                             40.75                                                                             18.32                                                                            12.98                                      5233-7                                                                            0.00                                                                             0.70                                                                              0.36                                                                             4.44                                                                              8.57                                                                             0.73                                                                              1.22                                                                             45.26                                                                             21.29                                                                            16.06                                      5233-8                                                                            0.00                                                                             1.07                                                                              0.24                                                                             2.46                                                                              7.67                                                                             0.85                                                                              1.25                                                                             47.47                                                                             22.51                                                                            14.90                                      5233-9                                                                            0.00                                                                             0.94                                                                              0.58                                                                             5.37                                                                              9.06                                                                             0.68                                                                              1.42                                                                             46.70                                                                             20.26                                                                            13.95                                      5233-10                                                                           0.00                                                                             0.83                                                                              0.26                                                                             2.84                                                                              7.89                                                                             0.74                                                                              1.26                                                                             46.21                                                                             21.68                                                                            16.88                                      5233-11                                                                           0.00                                                                             1.06                                                                              0.19                                                                             1.78                                                                              7.43                                                                             0.69                                                                              1.16                                                                             49.30                                                                             21.79                                                                            15.07                                      5233-12                                                                           0.00                                                                             0.69                                                                              0.51                                                                             5.42                                                                              9.02                                                                             0.77                                                                              1.40                                                                             46.09                                                                             19.81                                                                            15.03                                      5233-13                                                                           0.00                                                                             0.65                                                                              0.04                                                                             0.11                                                                              5.49                                                                             0.46                                                                              1.25                                                                             51.21                                                                             22.32                                                                            16.82                                      5233-14                                                                           0.00                                                                             0.81                                                                              0.64                                                                             6.46                                                                              9.54                                                                             0.86                                                                              1.21                                                                             44.11                                                                             20.50                                                                            14.33                                      5233-15                                                                           0.00                                                                             0.88                                                                              0.24                                                                             2.79                                                                              8.16                                                                             0.72                                                                              1.40                                                                             47.47                                                                             21.50                                                                            15.35                                      5233-16                                                                           0.00                                                                             1.00                                                                              0.35                                                                             3.52                                                                              8.03                                                                             0.66                                                                              1.35                                                                             44.98                                                                             23.01                                                                            15.94                                      5233-17                                                                           0.00                                                                             0.86                                                                              0.78                                                                             7.89                                                                              10.63                                                                            1.01                                                                              1.37                                                                             42.11                                                                             20.58                                                                            13.17                                      5233-18                                                                           0.00                                                                             1.53                                                                              0.62                                                                             6.25                                                                              10.14                                                                            0.83                                                                              1.37                                                                             39.80                                                                             23.38                                                                            15.64                                      5233-19                                                                           0.00                                                                             1.29                                                                              0.27                                                                             2.43                                                                              8.46                                                                             1.19                                                                              1.72                                                                             45.19                                                                             23.78                                                                            13.60                                      5233-20                                                                           0.00                                                                             1.23                                                                              0.34                                                                             3.59                                                                              9.49                                                                             1.03                                                                              1.87                                                                             48.74                                                                             19.24                                                                            12.93                                      5233-21                                                                           0.00                                                                             0.82                                                                              0.23                                                                             1.97                                                                              7.27                                                                             0.77                                                                              1.26                                                                             49.20                                                                             22.11                                                                            14.91                                      5233-22                                                                           0.00                                                                             0.60                                                                              0.54                                                                             5.63                                                                              9.64                                                                             0.75                                                                              1.56                                                                             45.07                                                                             21.94                                                                            12.53                                      5233-24                                                                           0.00                                                                             0.77                                                                              0.54                                                                             6.08                                                                              9.67                                                                             0.84                                                                              1.27                                                                             42.47                                                                             21.15                                                                            15.74                                      Control                                                                           0.00                                                                             0.74                                                                              0.02                                                                             0.10                                                                              6.15                                                                             0.66                                                                              1.48                                                                             51.75                                                                             21.03                                                                            16.22                                      __________________________________________________________________________

An increased percentage of C14 fatty acyl groups above control plantbackground levels is observed in all but one of the transgenic plantsanalyzed. The levels of C14 range from approximately 2.0 mole percent to11.5 mole percent. Increases in 16:0, and to a lesser extent, 12:0,fatty acyl groups are also observed.

Single seed data from transformants 5233-5 and 5233-6 are presented inFIG. 9. These results demonstrate C14 levels of greater than 20% areobtained in seeds expressing a camphor FatB thioesterase.

D. Elm Expression Construct

Mature seeds were harvested from transgenic Brassica napus plantscontaining pCGN4803, a construct for expression of elm thioesteraseclone ULM-1 under the regulatory control of a napin promoter, andanalyzed to determine mole percent fatty acid composition. Results arepresented in Table 8 below.

                                      TABLE 8                                     __________________________________________________________________________    Plant                                                                             8:0                                                                              10:0                                                                              12:0                                                                             14:0                                                                              16:0                                                                             16:1                                                                              18:0                                                                             18:1                                                                              18:2                                                                             18:3                                       __________________________________________________________________________    4803-8                                                                            0.00                                                                             1.60                                                                              0.43                                                                             5.13                                                                              24.31                                                                            0.97                                                                              3.89                                                                             34.00                                                                             19.33                                                                            7.75                                       4803-9                                                                            0.00                                                                             1.49                                                                              0.44                                                                             5.04                                                                              20.64                                                                            1.05                                                                              2.22                                                                             35.32                                                                             22.36                                                                            9.27                                       4803-10                                                                           0.00                                                                             3.05                                                                              1.19                                                                             10.87                                                                             28.97                                                                            0.58                                                                              2.52                                                                             25.91                                                                             15.19                                                                            9.92                                       4803-11                                                                           0.00                                                                             2.24                                                                              0.61                                                                             5.63                                                                              21.16                                                                            0.96                                                                              2.53                                                                             36.18                                                                             19.62                                                                            8.80                                       4803-12                                                                           0.00                                                                             4.06                                                                              1.53                                                                             13.44                                                                             32.24                                                                            0.67                                                                              3.01                                                                             20.80                                                                             14.85                                                                            7.21                                       4803-14                                                                           0.00                                                                             3.89                                                                              1.41                                                                             11.60                                                                             29.13                                                                            0.59                                                                              2.71                                                                             25.71                                                                             14.76                                                                            8.18                                       4803-15                                                                           0.00                                                                             2.92                                                                              0.85                                                                             7.82                                                                              26.29                                                                            0.90                                                                              2.98                                                                             27.69                                                                             19.99                                                                            8.00                                       4803-16                                                                           0.00                                                                             3.07                                                                              1.33                                                                             12.33                                                                             30.91                                                                            0.52                                                                              2.70                                                                             26.42                                                                             12.90                                                                            7.94                                       4803-17                                                                           0.00                                                                             1.63                                                                              0.42                                                                             5.51                                                                              24.62                                                                            0.77                                                                              2.31                                                                             36.23                                                                             16.50                                                                            9.79                                       4803-18                                                                           0.00                                                                             2.14                                                                              0.81                                                                             7.97                                                                              27.08                                                                            0.53                                                                              2.75                                                                             33.31                                                                             15.03                                                                            8.22                                       4803-19                                                                           0.00                                                                             2.76                                                                              0.63                                                                             4.99                                                                              18.58                                                                            0.92                                                                              2.61                                                                             39.61                                                                             15.55                                                                            11.83                                      4803-20                                                                           0.00                                                                             2.60                                                                              0.86                                                                             8.46                                                                              27.38                                                                            0.60                                                                              2.61                                                                             30.00                                                                             16.37                                                                            8.80                                       4803-21                                                                           0.00                                                                             1.27                                                                              0.40                                                                             4.95                                                                              20.51                                                                            0.55                                                                              2.10                                                                             39.72                                                                             16.98                                                                            11.38                                      4803-22                                                                           0.00                                                                             1.52                                                                              0.18                                                                             1.99                                                                              14.80                                                                            1.09                                                                              3.72                                                                             46.53                                                                             18.26                                                                            9.07                                       4803-23                                                                           0.00                                                                             2.65                                                                              0.45                                                                             3.65                                                                              18.78                                                                            0.88                                                                              3.17                                                                             41.45                                                                             17.37                                                                            8.99                                       4803-27                                                                           0.00                                                                             2.91                                                                              0.51                                                                             3.68                                                                              18.11                                                                            0.91                                                                              3.19                                                                             40.29                                                                             18.03                                                                            9.73                                       __________________________________________________________________________

An increased percentage of C14 fatty acyl groups above control plantbackground levels is observed in all of the transgenic plants analyzed.The levels of C14 range from approximately 2.0 mole percent to 13 molepercent. Increases in 16:0, and to a lesser extent, 10:0 and 12:0, fattyacyl groups are also observed.

E. C. palustris Expression Construct

Analysis of pooled seeds from four 3863-transformants reveals C14 levelsof approximately 25%. Data from analysis of fatty acid compositions ofsingle seeds from transformants 3863-10, 3863-7, 3863-4, 3863-8, 3863-2,and 3863-5 are presented in FIG. 10. These data indicate C14 levels ofgreater than 40% are obtained by expression of C. palustris FatB2thioesterase clone.

The above results demonstrate the ability to obtain DNA sequences whichencode thioesterase activities, which sequences may be expressed inplant seed cells for manipulation of seed oil fatty acid composition. Inthis manner production of significant levels of C14 fatty acids C14 maybe obtained. The novel seed oils so produced may find uses in industryas whole oils, or can be fractionated using methods known in theindustry to provide sources of the C14 fatty acids incorporated into theoil.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 17                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1581 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCTCTAATACGACTCACTATAGGGAAAGCTGGTACGCCTGCAGGTACCGGTCCGGAATTC60                CCGGGTCGACCCACGCGTCCGCTGAGTTTGCTGGTTACCATTTTCCCTGCGAACAAAC118                 ATGGTGGCTGCCGCAGCAAGTGCTGCATTCTTCTCCGTCGCAACCCCG166                           MetValAlaAlaAlaAlaSerAlaAlaPhePheSerValAlaThrPro                              151015                                                                        CGAACAAACATTTCGCCATCGAGCTTGAGCGTCCCCTTCAAGCCCAAA214                           ArgThrAsnIleSerProSerSerLeuSerValProPheLysProLys                              202530                                                                        TCAAACCACAATGGTGGCTTTCAGGTTAAGGCAAACGCCAGTGCCCAT262                           SerAsnHisAsnGlyGlyPheGlnValLysAlaAsnAlaSerAlaHis                              354045                                                                        CCTAAGGCTAACGGTTCTGCAGTAAGTCTAAAGTCTGGCAGCCTCGAG310                           ProLysAlaAsnGlySerAlaValSerLeuLysSerGlySerLeuGlu                              505560                                                                        ACTCAGGAGGACAAAACTTCATCGTCGTCCCCTCCTCCTCGGACTTTC358                           ThrGlnGluAspLysThrSerSerSerSerProProProArgThrPhe                              65707580                                                                      ATTAACCAGTTGCCCGTCTGGAGTATGCTTCTGTCTGCAGTCACGACT406                           IleAsnGlnLeuProValTrpSerMetLeuLeuSerAlaValThrThr                              859095                                                                        GTCTTCGGGGTGGCTGAGAAGCAGTGGCCAATGCTTGACCGGAAATCT454                           ValPheGlyValAlaGluLysGlnTrpProMetLeuAspArgLysSer                              100105110                                                                     AAGAGGCCCGACATGCTTGTGGAACCGCTTGGGGTTGACAGGATTGTT502                           LysArgProAspMetLeuValGluProLeuGlyValAspArgIleVal                              115120125                                                                     TATGATGGGGTTAGTTTCAGACAGAGTTTTTCGATTAGATCTTACGAA550                           TyrAspGlyValSerPheArgGlnSerPheSerIleArgSerTyrGlu                              130135140                                                                     ATAGGCGCTGATCGAACAGCCTCGATAGAGACCCTGATGAACATGTTC598                           IleGlyAlaAspArgThrAlaSerIleGluThrLeuMetAsnMetPhe                              145150155160                                                                  CAGGAAACATCTCTTAATCATTGTAAGATTATCGGTCTTCTCAATGAC646                           GlnGluThrSerLeuAsnHisCysLysIleIleGlyLeuLeuAsnAsp                              165170175                                                                     GGCTTTGGTCGAACTCCTGAGATGTGTAAGAGGGACCTCATTTGGGTG694                           GlyPheGlyArgThrProGluMetCysLysArgAspLeuIleTrpVal                              180185190                                                                     GTCACGAAAATGCAGATCGAGGTGAATCGCTATCCTACTTGGGGTGAT742                           ValThrLysMetGlnIleGluValAsnArgTyrProThrTrpGlyAsp                              195200205                                                                     ACTATAGAGGTCAATACTTGGGTCTCAGCGTCGGGGAAACACGGTATG790                           ThrIleGluValAsnThrTrpValSerAlaSerGlyLysHisGlyMet                              210215220                                                                     GGTCGAGATTGGCTGATAAGTGATTGCCATACAGGAGAAATTCTTATA838                           GlyArgAspTrpLeuIleSerAspCysHisThrGlyGluIleLeuIle                              225230235240                                                                  AGAGCAACGAGCGTGTGGGCTATGATGAATCAAAAGACGAGAAGATTG886                           ArgAlaThrSerValTrpAlaMetMetAsnGlnLysThrArgArgLeu                              245250255                                                                     TCGAAAATTCCATATGAGGTTCGACAGGAGATAGAGCCTCAGTTTGTG934                           SerLysIleProTyrGluValArgGlnGluIleGluProGlnPheVal                              260265270                                                                     GACTCTGCTCCTGTCATTGTAGACGATCGAAAATTTCACAAGCTTGAT982                           AspSerAlaProValIleValAspAspArgLysPheHisLysLeuAsp                              275280285                                                                     TTGAAGACCGGTGATTCCATTTGCAATGGTCTAACTCCAAGGTGGACT1030                          LeuLysThrGlyAspSerIleCysAsnGlyLeuThrProArgTrpThr                              290295300                                                                     GACTTGGATGTCAATCAGCACGTTAACAATGTGAAATACATCGGGTGG1078                          AspLeuAspValAsnGlnHisValAsnAsnValLysTyrIleGlyTrp                              305310315320                                                                  ATTCTCCAGAGTGTTCCCACAGAAGTTTTCGAGACGCAGGAGCTATGT1126                          IleLeuGlnSerValProThrGluValPheGluThrGlnGluLeuCys                              325330335                                                                     GGCCTCACCCTTGAGTATAGGCGAGAATGCGGAAGGGACAGTGTGCTG1174                          GlyLeuThrLeuGluTyrArgArgGluCysGlyArgAspSerValLeu                              340345350                                                                     GAGTCCGTGACCGCTATGGATCCATCAAAAGAGGGAGACCGGTCTCTT1222                          GluSerValThrAlaMetAspProSerLysGluGlyAspArgSerLeu                              355360365                                                                     TACCAGCACCTTCTCCGACTCGAGGACGGGGCTGATATCGTCAAGGGG1270                          TyrGlnHisLeuLeuArgLeuGluAspGlyAlaAspIleValLysGly                              370375380                                                                     AGAACCGAGTGGCGGCCGAAGAATGCAGGAGCCAAGGGAGCAATATTA1318                          ArgThrGluTrpArgProLysAsnAlaGlyAlaLysGlyAlaIleLeu                              385390395400                                                                  ACCGGAAAGACCTCAAATGGAAACTCTATATCTTAGAAGGAGGAAGGGACCTT1371                     ThrGlyLysThrSerAsnGlyAsnSerIleSer                                             405410                                                                        TCCGAGTTGTGTGTTTATTTGCTTTGCTTTGATTCACTCCATTGTATAATAATACTACGG1431              TCAGCCGTCTTTGTATTTGCTAAGACAAATAGCACAGTCATTAAGTTAAAAAAAAAAAAA1491              AAGGGCGGCCGCTCTAGAGGATCCAAGCTTACGTACGCGTGCATGCGACGTCATAGCTCT1551              TCTATAGTGTCACCTAAATTCAATTCACTG1581                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1360 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CCGGATTGGAGCATGCTTCTTGCAGCAATCACAACCATCTTCTTGGCA48                            ProAspTrpSerMetLeuLeuAlaAlaIleThrThrIlePheLeuAla                              151015                                                                        GCCGAGAAGCAGTGGACGAATCTTGACTGGAAGCCCAGGAGGCCTGAC96                            AlaGluLysGlnTrpThrAsnLeuAspTrpLysProArgArgProAsp                              202530                                                                        ATGCTCGTCGACTTTGACCCTTTTAGTCTGGGGAGGTTCGTTCAGGAT144                           MetLeuValAspPheAspProPheSerLeuGlyArgPheValGlnAsp                              354045                                                                        GGGTTGATTTTCAGGCAGAATTTCTCCATCAGGTCTTATGAGATTGGC192                           GlyLeuIlePheArgGlnAsnPheSerIleArgSerTyrGluIleGly                              505560                                                                        GCGGATCGGACGGCATCCATAGAGACGTTAATGAATCATCTACAGGAA240                           AlaAspArgThrAlaSerIleGluThrLeuMetAsnHisLeuGlnGlu                              65707580                                                                      ACGGCCCTAAACCATGTAAGGTGTATAGGGCTCCTCGATGATGGTTTT288                           ThrAlaLeuAsnHisValArgCysIleGlyLeuLeuAspAspGlyPhe                              859095                                                                        GGTTCGACGCCTGAGATGACTAGGAGAGATCTGATATGGGTGGTTACA336                           GlySerThrProGluMetThrArgArgAspLeuIleTrpValValThr                              100105110                                                                     AGGATGCAGGTTCTGGTGGATCGCTATCCTTCCTGGGGGGATGTCATT384                           ArgMetGlnValLeuValAspArgTyrProSerTrpGlyAspValIle                              115120125                                                                     GAAGTAGACTCCTGGGTTACTCCATCTGGAAAGAATGGGATGAAACGT432                           GluValAspSerTrpValThrProSerGlyLysAsnGlyMetLysArg                              130135140                                                                     GAATGGTTTCTCCGTGATTGCAAGACAGGCGAAATCCTGACACGAGCT480                           GluTrpPheLeuArgAspCysLysThrGlyGluIleLeuThrArgAla                              145150155160                                                                  ACCAGTGTTTGGGTGATGATGAATAAACGGACACGGAGGTTGTCCAAA528                           ThrSerValTrpValMetMetAsnLysArgThrArgArgLeuSerLys                              165170175                                                                     ATCCCTGAAGAAGTTAGAGTCGAAATAGAGCCTTATTTTGTGGAGCAT576                           IleProGluGluValArgValGluIleGluProTyrPheValGluHis                              180185190                                                                     GGAGTCTTGGATGAGGACAGCAGAAAACTACCAAAGCTCAATGACAAC624                           GlyValLeuAspGluAspSerArgLysLeuProLysLeuAsnAspAsn                              195200205                                                                     ACTGCAAATTACATCAGAAGAGGCCTAGCTCCTCGGTGGAGTGATTTA672                           ThrAlaAsnTyrIleArgArgGlyLeuAlaProArgTrpSerAspLeu                              210215220                                                                     GATGTCAATCAGCATGTGAACAATGTCAAATACATTGGCTGGATTCTT720                           AspValAsnGlnHisValAsnAsnValLysTyrIleGlyTrpIleLeu                              225230235240                                                                  GAGAGCGTGCCATCTTCACTGTTGGAGAGTCATGAGCTGTATGGGATG768                           GluSerValProSerSerLeuLeuGluSerHisGluLeuTyrGlyMet                              245250255                                                                     ACACTTGAGTATAGGAAGGAGTGTGGAAAGGACGGTTTGCTGCAATCC816                           LeuGluTyrArgLysGluCysGlyLysAspGlyLeuLeuGlnSer                                 260265270                                                                     CTGACTGCTGTTGCCAGTGATTATGGGGGTGGATCCCTTGAAGCTGGC864                           LeuThrAlaValAlaSerAspTyrGlyGlyGlySerLeuGluAlaGly                              275280285                                                                     GTTGAGTGTGACCACCTTCTTCGCCTTGAAGATGGGAGTGAGATTATG912                           ValGluCysAspHisLeuLeuArgLeuGluAspGlySerGluIleMet                              290295300                                                                     AGGGGAAAGACGGAATGGAGGCCCAAGCGTGCCGCCAACACTACCTAC960                           ArgGlyLysThrGluTrpArgProLysArgAlaAlaAsnThrThrTyr                              305310315320                                                                  TTTGGAAGCGTTGATGATATTCCTCCCCACCCAATATATATATATATA1008                          PheGlySerValAspAspIleProProHisProIleTyrIleTyrIle                              325330335                                                                     TATATATATATATATATATATATATATTGGGTGGGGAGCAGCTGCAGC1056                          TyrIleTyrIleTyrIleTyrIleTyrTrpValGlySerSerCysSer                              340345350                                                                     GGCAGCAGCACGACAATGTCGAGGACACGATGACGATCAGTATGTTTCGT1106                        GlySerSerThrThrMetSerArgThrArg                                                355360                                                                        GCGGTATTTAGCAATTCCGTATGTAGAATCCTGCGTGTACTGGCAGATAATTTTTTGATT1166              TGTTCTTTTCGTTTACGAGGGGAACCCGTGTAATTAGTTCAACTGTATTTTCTGTTTCTT1226              CCTTAAGTGTTTCAACACCCCTCTCTCTCTCGCGCGCGCGCGTGCGCTCACATTTTCCAT1286              TCCTTTTCTTTTTATTCTAGTTGTACGAGTGGGAGTTCATTTGCACTAAATTGTTGAAAA1346              ATCTCGTTGCTTGG1360                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1983 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGAGAGCCGCCTCTTCAGCCCACCACCACCTCTAAAACAACAGGCCCAAAACTCCCTCCT60                TTCTCTGTCCCTTTCCGGTGCTTCCCCCTCTATTTTAGACCTCCTCCTTTATATTTCCCA120               ACGTAGAATAATACCAAAACCCTAAACCGAGAAGAAGATAAAAGAAAGAGGAGAGAGAAA180               CAGAAAGAGATAGAGAGAGAAAAAAAATCGGTCTTCTCTCTCTTTCTCTGTCGCTGCGAA240               GGAGCGGCCGTGAAATTTGGTCATTTGCTATGAGAAATATTCCTTCTGTGATGCTTGATT300               TCTAATTTAACGAGTCTGTATCGTAATTTTCTCATCATGGTTGCCACATCTGCT354                     MetValAlaThrSerAla                                                            15                                                                            GCCTCCGCTTTCTTCCCGGTTGCCTCTCCGTCTCCAGTGAAGCCTTCG402                           AlaSerAlaPhePheProValAlaSerProSerProValLysProSer                              101520                                                                        ATGATGCTCGGTGGTGGAGGAGGTTCGGATAATCTCGACGCCCGTGGG450                           MetMetLeuGlyGlyGlyGlyGlySerAspAsnLeuAspAlaArgGly                              253035                                                                        ATCAAATCCCGCCCTGCCTCCTCTGGTGGCCTTCAAGTAAAGGCCAAT498                           IleLysSerArgProAlaSerSerGlyGlyLeuGlnValLysAlaAsn                              404550                                                                        GCTCATACTGTTCCCAAGATCAATGGTAACAAGGCGGGCCTTTTGACG546                           AlaHisThrValProLysIleAsnGlyAsnLysAlaGlyLeuLeuThr                              55606570                                                                      CCTATGGAGAGCACTAAGGACGAGGACATCGTGGCTGCCCCAACGGTT594                           ProMetGluSerThrLysAspGluAspIleValAlaAlaProThrVal                              758085                                                                        GCTCCTAAGAGGACTTTCATCAACCAGCTGCCGGATTGGAGCATGCTT642                           AlaProLysArgThrPheIleAsnGlnLeuProAspTrpSerMetLeu                              9095100                                                                       CTTGCAGCAATCACAACCATCTTCTTGGCAGCCGAGAAGCAGTGGACG690                           LeuAlaAlaIleThrThrIlePheLeuAlaAlaGluLysGlnTrpThr                              105110115                                                                     AATCTTGACTGGAAGCCCAGGAGGCCTGACATGCTCGTCGACTTTGAC738                           AsnLeuAspTrpLysProArgArgProAspMetLeuValAspPheAsp                              120125130                                                                     CCTTTTAGTCTGGGGAGGTTCGTTCAGGATGGGTTGATTTTCAGGCAG786                           ProPheSerLeuGlyArgPheValGlnAspGlyLeuIlePheArgGln                              135140145150                                                                  AATTTCTCCATCAGGTCTTATGAGATTGGCGCGGATCGGACGGCATCC834                           AsnPheSerIleArgSerTyrGluIleGlyAlaAspArgThrAlaSer                              155160165                                                                     ATAGAGACGTTAATGAATCATCTACAGGAAACGGCCCTAAACCATGTA882                           IleGluThrLeuMetAsnHisLeuGlnGluThrAlaLeuAsnHisVal                              170175180                                                                     AGGTGTATAGGGCTCCTCGATGATGGTTTTGGTTCGACGCCTGAGATG930                           ArgCysIleGlyLeuLeuAspAspGlyPheGlySerThrProGluMet                              185190195                                                                     ACTAGGAGAGATCTGATATGGGTGGTTACAAGGATGCAGGTTCTGGTG978                           ThrArgArgAspLeuIleTrpValValThrArgMetGlnValLeuVal                              200205210                                                                     GATCGCTATCCTTCCTGGGGGGATGTCATTGAAGTAGACTCCTGGGTT1026                          AspArgTyrProSerTrpGlyAspValIleGluValAspSerTrpVal                              215220225230                                                                  ACTCCATCTGGAAAGAATGGGATGAAACGTGAATGGTTTCTCCGTGAT1074                          ThrProSerGlyLysAsnGlyMetLysArgGluTrpPheLeuArgAsp                              235240245                                                                     TGCAAGACAGGCGAAATCCTGACACGAGCTACCAGTGTTTGGGTGATG1122                          CysLysThrGlyGluIleLeuThrArgAlaThrSerValTrpValMet                              250255260                                                                     ATGAATAAACGGACACGGAGGTTGTCCAAAATCCCTGAAGAAGTTAGA1170                          MetAsnLysArgThrArgArgLeuSerLysIleProGluGluValArg                              265270275                                                                     GTCGAAATAGAGCCTTATTTTGTGGAGCATGGAGTCTTGGATGAGGAC1218                          ValGluIleGluProTyrPheValGluHisGlyValLeuAspGluAsp                              280285290                                                                     AGCAGAAAACTACCAAAGCTCAATGACAACACTGCAAATTACATCAGA1266                          SerArgLysLeuProLysLeuAsnAspAsnThrAlaAsnTyrIleArg                              295300305310                                                                  AGAGGCCTAGCTCCTCGGTGGAGTGATTTAGATGTCAATCAGCATGTG1314                          ArgGlyLeuAlaProArgTrpSerAspLeuAspValAsnGlnHisVal                              315320325                                                                     AACAATGTCAAATACATTGGCTGGATTCTTGAGAGCGTGCCATCTTCA1362                          AsnAsnValLysTyrIleGlyTrpIleLeuGluSerValProSerSer                              330335340                                                                     CTGTTGGAGAGTCATGAGCTGTATGGGATGACACTTGAGTATAGGAAG1410                          LeuLeuGluSerHisGluLeuTyrGlyMetThrLeuGluTyrArgLys                              345350355                                                                     GAGTGTGGAAAGGACGGTTTGCTGCAATCCCTGACTGCTGTTGCCAGT1458                          GluCysGlyLysAspGlyLeuLeuGlnSerLeuThrAlaValAlaSer                              360365370                                                                     GATTATGGGGGTGGATCCCTTGAAGCTGGCGTTGAGTGTGACCACCTT1506                          AspTyrGlyGlyGlySerLeuGluAlaGlyValGluCysAspHisLeu                              375380385390                                                                  CTTCGCCTTGAAGATGGGAGTGAGATTATGAGGGGAAAGACGGAATGG1554                          LeuArgLeuGluAspGlySerGluIleMetArgGlyLysThrGluTrp                              395400405                                                                     AGGCCCAAGCGTGCCGCCAACACTACCTACTTTGGAAGCGTTGATGAT1602                          ArgProLysArgAlaAlaAsnThrThrTyrPheGlySerValAspAsp                              410415420                                                                     ATTCCTCCAGCAAATAATGCATAGCCAAAATGTATATATATATATATATAT1653                       IleProProAlaAsnAsnAla                                                         425                                                                           ATATATATATATATATATATATATATATATATTGGGTGGGGAGCAGCTGCAGCGGCAGCA1713              GCACGACAATGTCGAGGACACGATGACGATCAGTATGTTTCGTGCGGTATTTAGCAATTC1773              CGTATGTAGAATCCTGCGTGTACTGGCAGATAATTTTTTGATTTGTTCTTTTCGTTTACG1833              AGGGGAACCCGTGTAATTAGTTCAACTGTATTTTCTGTTTCTTCCTTAAGTGTTTCAACA1893              CCCCTCTCTCTCTCGCGCGCGCGCGTGCGCTCACATTTTCCATTCCTTTTCTTTTTATTC1953              TAGTTGTACGAGTGGGAGTTCATTTGCACT1983                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1157 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TCTAGAGTGGAAGCCGAAGCCGAATCCACCCCAGTTGCTTGATGACCAT49                           LeuGluTrpLysProLysProAsnProProGlnLeuLeuAspAspHis                              151015                                                                        TTTGGGCCGCATGGGTTAGTTTTCAGGCGCACCTTTGCCATCAGATCG97                            PheGlyProHisGlyLeuValPheArgArgThrPheAlaIleArgSer                              202530                                                                        TATGAGGTGGGACCTGACCGCTCCACATCTATAGTGGCTGTTATGAAT145                           TyrGluValGlyProAspArgSerThrSerIleValAlaValMetAsn                              354045                                                                        CACTTGCAGGAGGCTGCACTTAATCATGCGAAGAGTGTGGGAATTCTA193                           HisLeuGlnGluAlaAlaLeuAsnHisAlaLysSerValGlyIleLeu                              505560                                                                        GGAGATGGATTCGGTACGACGCTAGAGATGAGTAAGAGAGATCTGATA241                           GlyAspGlyPheGlyThrThrLeuGluMetSerLysArgAspLeuIle                              65707580                                                                      TGGGTTGTGAAACGCACGCATGTTGCTGTGGAACGGTACCCTGCTTGG289                           TrpValValLysArgThrHisValAlaValGluArgTyrProAlaTrp                              859095                                                                        GGTGATACTGTTGAAGTAGAGTGCTGGGTTGGTGCATCGGGAAATAAT337                           GlyAspThrValGluValGluCysTrpValGlyAlaSerGlyAsnAsn                              100105110                                                                     GGCAGGCGCCATGATTTCCTTGTCCGGGACTGCAAAACAGGCGAAATT385                           GlyArgArgHisAspPheLeuValArgAspCysLysThrGlyGluIle                              115120125                                                                     CTTACAAGATGTACCAGTCTTTCGGTGATGATGAATACAAGGACAAGG433                           LeuThrArgCysThrSerLeuSerValMetMetAsnThrArgThrArg                              130135140                                                                     AGGTTGTCCAAAATCCCTGAAGAAGTTAGAGGGGAGATAGGGCCTGCA481                           ArgLeuSerLysIleProGluGluValArgGlyGluIleGlyProAla                              145150155160                                                                  TTCATTGATAATGTGGCTGTCAAAGACGAGGAAATTAAGAAACCACAG529                           PheIleAspAsnValAlaValLysAspGluGluIleLysLysProGln                              165170175                                                                     AAGCTCAATGACAGCACTGCAGATTACATCCAAGGAGGATTGACTCCT577                           LysLeuAsnAspSerThrAlaAspTyrIleGlnGlyGlyLeuThrPro                              180185190                                                                     CGATGGAATGATTTGGATATCAATCAGCACGTTAACAACATCAAATAC625                           ArgTrpAsnAspLeuAspIleAsnGlnHisValAsnAsnIleLysTyr                              195200205                                                                     GTTGACTGGATTCTTGAGACTGTCCCAGACTCAATCTTTGAGAGTCAT673                           ValAspTrpIleLeuGluThrValProAspSerIlePheGluSerHis                              210215220                                                                     CATATTTCCAGCTTCACTATTGAATACAGGAGAGAGTGCACGAGGGAT721                           HisIleSerSerPheThrIleGluTyrArgArgGluCysThrArgAsp                              225230235240                                                                  AGCGTGCTGCAGTCCCTGACCACTGTCTCCGGTGGCTCGTCGGAAGCT769                           SerValLeuGlnSerLeuThrThrValSerGlyGlySerSerGluAla                              245250255                                                                     GGGTTAGTGTGCGAGCACTTGCTCCAGCTTGAAGGTGGGTCTGAGGTA817                           GlyLeuValCysGluHisLeuLeuGlnLeuGluGlyGlySerGluVal                              260265270                                                                     TTGAGGGCAAAAACAGAGTGGAGGCCTAAGCTTACCGATAGTTTCAGA865                           LeuArgAlaLysThrGluTrpArgProLysLeuThrAspSerPheArg                              275280285                                                                     GGGATTAGTGTGATACCCGCAGAATCGAGTGTCTAACTAACGAAAGAAGCATC918                      GlyIleSerValIleProAlaGluSerSerVal                                             290295                                                                        TGATGAAGTTTCTCCTGTGCTGTTGTTCGTGAGGATGCTTTTTAGAAGCTGCAGTTTGCA978               TTGCTTGTGCAGAATCATGGCCTGTGGTTTTAGATATATATTCAAAATTGTCCTATAGTC1038              AAGAAACTTAATATCAGAAAAATAACTCAATGAGTCAAGGTTATCGAAGTAGTCATGTAA1098              GCTTTGAAATATGTTGTGTATTCCTCGGCTTTATGTAATCTGTAAGCTCTTTCTCTTGC1157               (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1433 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GAATTCGGCACGAGGGGCTCCGGTGCTTTGCAGGTGAAGGCAAGTTCC48                            GluPheGlyThrArgGlySerGlyAlaLeuGlnValLysAlaSerSer                              51015                                                                         CAAGCTCCACCAAAGCTCAATGGTTCCAATGTGGGTTTGGTTAAATCT96                            GlnAlaProProLysLeuAsnGlySerAsnValGlyLeuValLysSer                              202530                                                                        AGCCAAATTGTGAAGAAGGGTGATGACACCACATCTCCTCCTGCAAGA144                           SerGlnIleValLysLysGlyAspAspThrThrSerProProAlaArg                              354045                                                                        ACTTTCATCAACCAATTGCCTGATTGGAGCATGCTTCTTGCTGCTATC192                           ThrPheIleAsnGlnLeuProAspTrpSerMetLeuLeuAlaAlaIle                              505560                                                                        ACAACCCTGTTCTTGGCTGCAGAGAAGCAGTGGATGATGCTTGATTGG240                           ThrThrLeuPheLeuAlaAlaGluLysGlnTrpMetMetLeuAspTrp                              65707580                                                                      AAACCCAAAAGGCCTGACATGCTTGTTGATCCATTTGGTCTTGGAAGG288                           LysProLysArgProAspMetLeuValAspProPheGlyLeuGlyArg                              859095                                                                        TTTGTTCAGGATGGTCTTGTTTTCCGCAACAACTTTTCAATTCGATCA336                           PheValGlnAspGlyLeuValPheArgAsnAsnPheSerIleArgSer                              100105110                                                                     TATGAAATAGGGGCTGATCGAACGGCTTCTATAGAAACGTTAATGAAT384                           TyrGluIleGlyAlaAspArgThrAlaSerIleGluThrLeuMetAsn                              115120125                                                                     CATCTGCAGGAAACAGCTCTTAATCATGTGAAGTCTGTTGGGCTTCTT432                           HisLeuGlnGluThrAlaLeuAsnHisValLysSerValGlyLeuLeu                              130135140                                                                     GAGGATGGCCTAGGTTCGACTCGAGAGATGTCCTTGAGGAACCTGATA480                           GluAspGlyLeuGlySerThrArgGluMetSerLeuArgAsnLeuIle                              145150155160                                                                  TGGGTTGTCACTAAAATGCAGGTTGCGGTTGATCGCTATCCAACTTGG528                           TrpValValThrLysMetGlnValAlaValAspArgTyrProThrTrp                              165170175                                                                     GGAGATGAAGTTCAGGTATCCTCTTGGGCTACTGCAATTGGAAAGAAT576                           GlyAspGluValGlnValSerSerTrpAlaThrAlaIleGlyLysAsn                              180185190                                                                     GGAATGCGTCGCGAATGGATAGTCACTGATTTTAGAACTGGTGAAACT624                           GlyMetArgArgGluTrpIleValThrAspPheArgThrGlyGluThr                              195200205                                                                     CTATTAAGAGCCACCAGTGTTTGGGTGATGATGAATAAACTGACGAGG672                           LeuLeuArgAlaThrSerValTrpValMetMetAsnLysLeuThrArg                              210215220                                                                     AGGATATCCAAAATCCCAGAAGAGGTTTGGCACGAAATAGGCCCCTCT720                           ArgIleSerLysIleProGluGluValTrpHisGluIleGlyProSer                              225230235240                                                                  TTCATTGATGCTCCTCCTCTTCCCACCGTGGAAGATGATGGTAGAAAG768                           PheIleAspAlaProProLeuProThrValGluAspAspGlyArgLys                              245250255                                                                     CTGACAAGGTTTGATGAAAGTTCTGCAGACTTTATCCGCNCTGGTTTA816                           LeuThrArgPheAspGluSerSerAlaAspPheIleArgXxxGlyLeu                              260265270                                                                     ACTCCTAGGTGGAGTGATTTGGACATCAACCAGCATGTCAACAATGTG864                           ThrProArgTrpSerAspLeuAspIleAsnGlnHisValAsnAsnVal                              275280285                                                                     AAGTACATTGGCTGGCTCCTTGAGAGTGCTCCGCCGGAGATCCACGAG912                           LysTyrIleGlyTrpLeuLeuGluSerAlaProProGluIleHisGlu                              290295300                                                                     AGTCACGAGATAGCGTCTCTGACTCTGGAGTACAGGAGGGAGTGTGGA960                           SerHisGluIleAlaSerLeuThrLeuGluTyrArgArgGluCysGly                              305310315320                                                                  AGGGACAGCGTGCTGAACTCCGCGACCAAGGTCTCTGACTCCTCTCAA1008                          ArgAspSerValLeuAsnSerAlaThrLysValSerAspSerSerGln                              325330335                                                                     CTGGGAAAGTCTGCTGTGGAGTGTAACCACTTGGTTCGTCTCCAGAAT1056                          LeuGlyLysSerAlaValGluCysAsnHisLeuValArgLeuGlnAsn                              340345350                                                                     GGTGGGGAGATTGTGAAGGGAAGGACTGTGTGGAGGCCCAAACGTCCT1104                          GlyGlyGluIleValLysGlyArgThrValTrpArgProLysArgPro                              355360365                                                                     CTTTACAATGATGGTGCTGTTGTGGACGTGNAAGCTAAAACCTCT1149                             LeuTyrAsnAspGlyAlaValValAspValXxxAlaLysThrSer                                 370375380                                                                     TAAGTCTTATAGTCCAAGTGAGGAGGAGTTCTATGTATCAGGAAGTTGCTAGGATTCTCA1209              ATCGCATGTGTCCATTTCTTGTGTGGAATACTGCTCGTGTTTCTAGACTCGCTATATGTT1269              TGTTCTTTTATATATATATATATATATATATCTCTCTCTTCCCCCCACCTCTCTCTCTCT1329              CTCTATATATATATATGTTTTATGTAAGTTTTCCCCTTAGTTTCCTTTCCTAAGTAATGC1389              CATTGTAAATTACTTCAAAAAAAAAAAAAAAAAAAAAACTCGAG1433                              (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 976 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GGCACGAGAAACATGGTGGCTGCCGCAGCAAGTTCTGCATTCTTCTCCGTTCCAACCCCG60                GGAATCTCCCCTAAACCCGGGAAGTTCGGTAATGGTGGCTTTCAGGTTAAGGCAAACGCC120               AATGCCCATCCTAGTCTAAAGTCTGGCAGCCTCGAGACTGAAGATGACACTTCATCGTCG180               TCCCCTCCTCCTCGGACTTTCATTAACCAGTTGCCCGACTGGAGTATGCTTCTGTCCGCA240               ATCACGACTATCTTCGGGGCAGCTGAGAAGCAGTGGATGATGCTTGATAGGAAATCTAAG300               NAGACCCGACATGCTCATGGCAACCGTTTGGGGTTGACAGTATTGTTCAGGATGGGGTTT360               TTTTCAGACAGAGTTTTTCGATTAGATCTTACGAAATAGGCGCTGATCGAACAACCTCAA420               TAGAGACGCTGATGAACATGTTCCAGGAAACGTCTTTGAATCATTGTAAGAGTAACGGTC480               TTCTCAATGACGGCTTTGGTCGCACTCCTGAGATGTGTAAGAAGGGCCTCATTTGGGTGG540               TTACGAAAATGCAGGTCGAGGTGAATCGCTATCCTATTTGGSGTGATTCTATCGAAGTCA600               ATACTTGGGTCTCCGAGTCGGGGNAAAANCGGTATGGGTCGTGATTGGCTGATAAGTGAT660               TGCAGTACAGGAGNAAATTCTTGTAAGAGCAACGAGCGTGTGGGCTATGATGAATCAAAA720               GACGAGAAGATTGTCAAAATTTCCATTTGAGGTTCGACAAGAGATAGCGCCTAATTTTGT780               CGACTCTGTTCCTGTCATTGAAGACGATCGAAAATTACACAAGCTTGATGTGAAGACGGG840               TGATTCCATTCACAATGGTCTAACTCCAAGGTGGAATGACTTGGATGTCAATCAGCACGT900               TAACAATGTGAAATACATTGGGTGGATTCTCAAGAGTGTTCCAACAGATGTTTTTGGGGC960               CCAGGAGCTATGTGGA976                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1647 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GGCGCGCCGGTACCTCTAGACCTGGCGATTCAACGTGGTCGGATCATGACGCTTCCAGAA60                AACATCGAGCAAGCTCTCAAAGCTGACCTCTTTCGGATCGTACTGAACCCGAACAATCTC120               GTTATGTCCCGTCGTCTCCGAACAGACATCCTCGTAGCTCGGATTATCGACGAATCCATG180               GCTATACCCAACCTCCGTCTTCGTCACGCCTGGAACCCTCTGGTACGCCAATTCCGCTCC240               CCAGAAGCAACCGGCGCCGAATTGCGCGAATTGCTGACCTGGAGACGGAACATCGTCGTC300               GGGTCCTTGCGCGATTGCGGCGGAAGCCGGGTCGGGTTGGGGACGAGACCCGAATCCGAG360               CCTGGTGAAGAGGTTGTTCATCGGAGATTTATAGACGGAGATGGATCGAGCGGTTTTGGG420               GAAAGGGGAAGTGGGTTTGGCTCTTTTGGATAGAGAGAGTGCAGCTTTGGAGAGAGACTG480               GAGAGGTTTAGAGAGAGACGCGGCGGATATTACCGGAGGAGAGGCGACGAGAGATAGCAT540               TATCGAAGGGGAGGGAGAAAGAGTGACGTGGAGAAATAAGAAACCGTTAAGAGTCGGATA600               TTTATCATATTAAAAGCCCAATGGGCCTGAACCCATTTAAACAAGACAGATAAATGGGCC660               GTGTGTTAAGTTAACAGAGTGTTAACGTTCGGTTTCAAATGCCAACGCCATAGGAACAAA720               ACAAACGTGTCCTCAAGTAAACCCCTGCCGTTTACACCTCAATGGCTGCATGGTGAAGCC780               ATTAACACGTGGCGTAGGATGCATGACGACGCCATTGACACCTGACTCTCTTCCCTTCTC840               TTCATATATCTCTAATCAATTCAACTACTCATTGTCATAGCTATTCGGAAAATACATACA900               CATCCTTTTCTCTTCGATCTCTCTCAATTCACAAGAAGCAAAGTCGACGGATCCCTGCAG960               TAAATTACGCCATGACTATTTTCATAGTCCAATAAGGCTGATGTCGGGAGTCCAGTTTAT1020              GAGCAATAAGGTGTTTAGAATTTGATCAATGTTTATAATAAAAGGGGGAAGATGATATCA1080              CAGTCTTTTGTTCTTTTTGGCTTTTGTTAAATTTGTGTGTTTCTATTTGTAAACCTCCTG1140              TATATGTTGTACTTCTTTCCCTTTTTAAGTGGTATCGTCTATATGGTAAAACGTTATGTT1200              TGGTCTTTCCTTTTCTCTGTTTAGGATAAAAAGACTGCATGTTTTATCTTTAGTTATATT1260              ATGTTGAGTAAATGAACTTTCATAGATCTGGTTCCGTAGAGTAGACTAGCAGCCGAGCTG1320              AGCTGAACTGAACAGCTGGCAATGTGAACACTGGATGCAAGATCAGATGTGAAGATCTCT1380              AATATGGTGGTGGGATTGAACATATCGTGTCTATATTTTTGTTGGCATTAAGCTCTTAAC1440              ATAGATATAACTGATGCAGTCATTGGTTCATACACATATATAGTAAGGAATTACAATGGC1500              AACCCAAACTTCAAAAACAGTAGGCCACCTGAATTGCCTTATCGAATAAGAGTTTGTTTC1560              CCCCCACTTCATGGGATGTAATACATGGGATTTGGGAGTTTGAATGAACGTTGAGACATG1620              GCAGAACCTCTAGAGGTACCGGCGCGC1647                                               (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: synthetic oligonucleotide                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CUACUACUACUATCGATACCATCTTTTCGGCTGCTGA37                                       (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: synthetic oligonucleotide                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CAUCAUCAUCAUGAGCTCGCAAGAGAAAGAGCTTACAG38                                      (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: synthetic oligonucleotide                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CUACUACUACUAGAATTCGCATGCAGGCCTATGCTTGACCGGAAATCT48                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: synthetic oligonucleotide                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GTTTTCCCAGTCACGAC17                                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: synthetic oligonucleotide                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CAUCAUCAUCAUGTCGACAAACATGGTGGCTGCCGCAG38                                      (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: synthetic oligonucleotide                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CUACUACUACUAATGCATTACTAAGATATAGAGTTTCCATTTG43                                 (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 100 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: synthetic oligonucleotide                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CGGTCTAGATAACAATCAATGCAAGACTATTGCACACGTGTTGCGTGTGAACAATGGTCA60                GGAGCTTCACGTCTGGGAAACGCCCCCAAAAGAAAACGTG100                                   (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 100 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: synthetic oligonucleotide                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ATACTCGGCCAATCCAGCGAAGTGGTCCATTCTTCTGGCGAAACCAGAAGCAATCAAAAT60                GGTGTTGTTTTTAAAAGGCACGTTTTCTTTTGGGGGCGTT100                                   (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: synthetic oligonucleotide                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CAUCAUCAUCAUGGATCCCTCATCATGGTTGCCACATCTGC41                                   (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: synthetic oligonucleotide                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CUACUACUACUACTCGAGTTACATTTTGGCTATGC35                                         __________________________________________________________________________

What is claimed is:
 1. A method of producing C14 fatty acids in plantseed triglycerides, wherein said method comprises:growing a plant havingintegrated into its genome a DNA construct, said construct comprising inthe 5' to 3' direction of transcription, a promoter functional in aplant seed cell, a DNA sequence encoding a plant thioesterase proteinwhich preferentially utilizes C14:0 acyl-ACP substrates as compared toother medium-chain acyl-ACP substrates or as compared to C16:0 acyl-ACPsubstrates, and a transcription termination region functional in a plantcell, wherein said DNA sequence is obtained from camphor or Cupheapalustris.
 2. The method of claim 1 wherein said plant is an oilseedcrop plant.
 3. The method of claim 2 wherein said oilseed crop plant isa Brassica plant.
 4. The method of claim 1, wherein said promoter isfrom a gene preferentially expressed in plant seed tissue.
 5. The methodof claim 1, wherein said plant seed triglycerides comprise at least 5mole percent C14 fatty acyl groups.
 6. The method of claim 1, whereinsaid plant seed triglycerides comprise at least 20 mole percent C14fatty acyl groups.
 7. The method of claim 1, wherein said plant seedtriglycerides comprise at least 40 mole percent C14 fatty acyl groups.8. A plant seed comprising a minimum of 40 mole percent myristate intotal fatty acids, wherein said seed has been transformed to express aCuphea palustris DNA sequence encoding a thioesterase protein whichpreferentially utilizes C14:0 acyl-ACP substrates as compared to othermedium-chain acyl-ACP substrates or as compared to C16:0 acyl-ACPsubstrates, wherein said myristate is incorporated into at least oneposition of a triglyceride molecule and wherein wild-type seed of saidplant contains less than 1.0 mole percent laurate in fatty acids. 9.Plant seed oil, wherein a minimum of 40 mole percent of the acyl groupsof said oil are myristyl acyl groups, and wherein said oil is derivedfrom a seed of claim
 8. 10. A Brassica seed comprising a minimum of 40mole percent myristate in total fatty acids, wherein said seed has beentransformed to express a Cuphea palustris DNA sequence encoding athioesterase protein.
 11. Plant seed oil, wherein a minimum of 40 molepercent of the acyl groups of said oil are myristyl acyl groups, andwherein said oil is derived from a Brassica seed of claim
 10. 12. A DNAconstruct comprising an encoding sequence for a plant acyl-ACPthioesterase which preferentially utilizes C14 acyl-ACP substrates ascompared to other medium-chain acyl-ACP substrates or as compared toC16:0 acyl-ACP substrates, wherein said plant is Cuphea palustris.
 13. Aconstruct according to claim 12 wherein said thioesterase comprises theamino acid sequence shown in FIG.
 1. 14. A construct according to claim13 wherein said encoding sequence comprises the acyl-ACP thioesteraseencoding sequence shown in FIG. 1.